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Why are there two replicase proteins translated from tobacco mosaic virus RNA?

Why are there two replicase proteins translated from tobacco mosaic virus RNA?


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I'm trying to understand how TMV is expressed and have read (here) that there is a large and small form of the RNA-dependent RNA replicase. These are translated from the same region of the genome, the larger arising from read-through of a leaky stop codon.

Why are there two forms of the replicase? My first impression was that each might produce a sub-genomic RNA, one for the movement protein and one for the capsid protein, but can find no reports confirming this.


Short Answer

As of 2021 the rationale for the production of two proteins from the Tobacco Mosaic Virus (TMV) replicase gene is incompletely understood. The two proteins share some activities but not others, and although they appear to co-operate in the replication process there is a ten-fold excess of the smaller relative to the larger.

Longer Answer

“Why is there a 126-kDa and 183-kDa form of the TMV RNA-dependent RNA replicase?” My answer is based mainly on the review by Buck (Phil. Trans. R. Soc. Lond. B (1999) 354, 613-627), but includes material from some later sources (e.g. Malpica-López et al. (2018)). Many relatively recent papers related to the replicase (e.g. this from 2008, and this from 2012 tend to avoid this question, confining themselves to describing the properties of the two proteins. The main points are:

  • Both the TMV-encoded 126-kDa and 183-kDa proteins are required for maximum efficiency of TMV RNA replication. The full-length protein is absolutely required; preventing the production of the smaller protein reduces the replication rate to 20% of normal.
  • The rate of read-through of the stop codon is about 10%, so that ratio of 126-kDa :183-kDa proteins is 10:1.
  • The read-through portion of the 183-kDa protein contains amino-acid motifs characteristic of RNA- dependent RNA polymerases (RdRps) and hence the 183-kDa protein is likely to provide the catalytic activity for the synthesis of TMV RNA from NTP substrates.
  • “The C-terminal domain of the TMV 126-kDa protein is helicase-like. Although helicase activity has not yet been demonstrated for the 126-kDa protein of any tobamovirus, these proteins contain six amino-acid motifs, which are highly conserved in known helicases, such as the translational initiation factor eIF-4A.”
  • “The TMV 126-kDa protein contains two domains. An N-terminal domain with amino-acid motifs and predicted secondary structure typical of S-adenosylmethionine binding proteins, methyltransferases and guanylyltransferases is probably required for synthesis of the 5' m7GpppG cap structure. The TMV 126-kDa protein has been shown to have guanylyltransferase activity.”
  • The 126-kDa protein has activity in silencing host anti-virus suppression. The 183-kDa protein lacks this activity, suggesting a difference in conformation relating to the region responsible.
  • There is evidence for association of two forms of replicase in the replication of membrane-bound viral RNA. However no direct structural information (e.g. X-ray crystallography, cryo-EM) is available for a complex with or without the RNA.

The only suggestion that Buck makes is that as helicase activity is required for two separate purposes - to unwind double-stranded RNA during replication, and to remove secondary structure from single-stranded RNA when this is used as a template for multiple copies of the complementary strand - the two forms of the replicase may have a different substrate specificity in their helicase activity.

Own thoughts

It is commonplace that the constraints on eukaryotic mRNA translational initiation and the small size of viral genomes have resulted in viruses adopting different strategies for maximizing their coding potential. Read-through is not unique to this class of viruses. I imagine that the replicase preceded the readthrough, in which case one wonders whether the original enzyme was a homo-dimer, which has been superseded by a hetero-dimer with similar protein-protein interaction. The problem here is that the contemporary enzyme has only one active site. Clearly, structural information is needed to address these possibilities.

Footnote: Nomenclature

The original question and some sources (mainly in structural biology) refer to the 126-kDa and 183-kDa forms as subunits of the replicase. Most virological sources do not employ this description, which I think is misleading because it ignores 90% of 126-kDa protein which cannot be involved in replication and therefore does not function as a replicase subunit. It also suggests a level of structural analysis that does not exist. Although replicase enzymes often have several subunits, they are generally structurally distinct. The situation with TMV is different.


The RNA-dependent RNA polymerase of the Tobamoviruses exists as a heterodimer expressed by read-through of the RdRP portion of the genome (open reading frames 1 and 2). The products of these ORFs are two proteins of 183 kDa (large replicase subunit) and 126 kDa (small replicase subunit), with the 126 kDa being produced approximately 10x more than the 183 kDa form. The functions and sizes of the various proteins are described well in Watanabe et al., (1999):

The genome of tobacco mosaic virus (TMV) consists of a single-stranded RNA molecule of about 6,400 nucleotides in length with positive polarity, which encodes at least four polypeptides: 126- and 183-kDa proteins required for transcription and replication (hereafter referred to as the 126K and 183K proteins, respectively), a 30-kDa (30K) protein for cell-to-cell virus movement in infected plants, and an 18-kDa protein for virus coat formation. The sequence of the 126K protein is encoded by the 5'-proximal region of the viral genome and includes the methyltransferase and RNA helicase motifs, while the 183K protein is a read-through protein of the 126K open reading frame (ORF) and contains, in addition to the above two motifs, the RNA-dependent RNA polymerase motif. The RNA polymerase is considered to be involved in both transcription and replication (8). From sequence analysis, it is believed that the viral RNA polymerase contains the 183K protein as a catalytic subunit, but the precise molecular compositions of transcriptase and replicase have not yet been determined.

A common feature of RdRPs in RNA viruses is that they exist as heteromers. A very well known example of this being the RdRP of influenza virus (-ssRNA), which exists as a heterotrimer consisting of the PB1, PB2 and PA subunits produced from the eponymous genome fragments. However, (many) other examples exist in the virus world, including in another plant virus genus Potyvirus, which, in common with Tobamoviruses and 90% of plant viruses, is a positive sense single-stranded RNA virus. The features of the Potyvirus RdRP have just been published, and to quote the linked article (and references therein):

The RdRp-RdRp self-interaction seems to be a common feature for positive-sense, single-stranded RNA viruses, including insect-, animal- and human-, and plant-infecting viruses [38,49,50,51,52,53]. The dimerization or oligomerization of RdRps may increase the stability of these enzymes and protect against degradation.

As you can see from the information provided in the top quote, the functions of the different subunits of the protein are different but complementary. The 126 kDa contains a helicase and methyltransferase motifs, while the 183 kDa functions as the polymerase as well as containing the same helicase and methyltransferase motifs. Lewandowski and Dawson (2000) found that the 183 kDa subunit was capable of performing all the functions listed above, acting as the full RdRP, but with the 126 kDa subunit those functions were performed ~10 times faster. They also found that mutating a base in the helicase domain of the 126 kDa (183 kDa supplied by a helper virus that only expresses the 183 kDa form) resulted in no replication of the RNA with the mutant 126 kDa indicating that this protein is essential to RNA replication. It should also be noted here as per comments, that the evidence for heterodimerism of the two forms is not in the form of a crystal structure, but in a 1:1 stoichiometry in immunoprecipitation as discussed in the Watanabe paper linked above. To my knowledge no-one has produced a full crystal structure of the RdRP of any Tobamovirus.

However, as you might have noticed when you looked at the information on the link you provided, that there seemed to be only about 4 ORFs, and a small number of proteins produced, and you might be thinking something along the lines of

"how does a virus manage to work when it only produces so few proteins?"

The answer to which is viral proteins perform many functions (this feature isn't specific to viral proteins). In particular, the small subunit (126 kDa) seems to act as a suppressor of the host silencing RNA system (siRNA). siRNA systems in plants function a bit like an immune system response - they signal within the cell and extra-cellularly to induce a viral RNA degrading response, so that the virus can not easily spread or establish itself in the host. I would speculate that what better place to suppress siRNA functions than at the site of viral RNA replication itself.


Virus-specific capping of tobacco mosaic virus RNA: methylation of GTP prior to formation of covalent complex p126-m 7 GMP

In capping cellular mRNAs, a covalent GMP-enzyme intermediate leads to formation of G(5′)ppp(5′)N at the 5′ end of the RNA, which is modified by methylation catalyzed by guanine-7-methyltransferase. Here we show that isolated membranes from tobacco mosaic virus (TMV)-infected plant or insect cells expressing TMV replicase protein p126, synthesized m 7 GTP using S-adenosylmethionine (AdoMet) as the methyl donor, and catalyzed the formation of a covalent guanylate-p126 complex in the presence of AdoMet. The methyl group from AdoMet was incorporated into p126, suggesting that the complex consisted of m 7 GMP-p126. Thus, TMV and alphaviruses, despite their evolutionary distance, share the same virus-specific capping mechanism.


Introduction

Viruses, as obligate organisms, utilize host factors to accumulate and spread in their host. A successful infection by a plant virus includes entry and accumulation in the first cell, movement into neighboring uninfected cells, and systemic infection through the plant vascular tissue (Boevink and Oparka, 2005 Epel, 2009 Harries and Ding, 2011 Niehl and Heinlein, 2011 Schoelz et al., 2011 Tilsner et al., 2011). Plant viruses have varying strategies for infecting hosts which reflect their use of existing functionally redundant host developmental pathways. Therefore an understanding of virus infection processes also offers insight into normal host physiological processes. Tobacco mosaic virus (TMV) encodes four known functional proteins: the 126 and 183 kDa replication-associated proteins, the movement protein (MP), and the structural capsid or coat protein (CP). In order to have a successful infection, these four multifunctional proteins cooperate with many host components. The host membrane and cytoskeleton are sub-cellular structures important for TMV infection. TMV-induced granules or inclusion bodies that contain membranes also contain host proteins. In this review, we discuss the changing roles of host membranes, cytoskeleton, and inclusion body-associated proteins as infection progresses. Findings reported in the literature are first presented in the section(s) where the effect on virus physiology was observed rather than where it may additionally influence this activity. For example, the influence of synaptotagmin on TMV physiology (Lewis and Lazarowitz, 2010) was reported as an inhibition of intercellular spread of the TMV MP, although it likely influences the intracellular transport of this protein. This was done to clearly indicate what is in the published literature rather than what a reader may interpret the results to indicate. In some instances, however, the presumed influence of the observed outcome on the mechanism of virus movement is noted. As pertinent, findings from other tobamoviruses are mentioned to indicate the generality or specificity of a conclusion for the genus.


Virus Genetics and Evolution

As Yoshimi Okada (Teikyo University, Utsunomiya, Japan) reminded those attending the symposium, it was not until the 1950s that most biologists accepted that genes are constructed from nucleic acids. Experiments with TMV played an important role in this development by providing the first unequivocal demonstration that a viral RNA molecule—specifically the TMV RNA—was sufficient for infectivity and carried all of the information necessary for synthesis of the CP ( Fraenkel-Conrat, 1956 Gierer and Schramm, 1956).

Bea Singer (University of California, Berkeley) recounted how she and H. Fraenkel-Conrat (University of California, Berkeley) extended biochemical research of TMV genetics. They began with naturally occurring TMV strains to demonstrate that the progeny of mixed viruses (i.e., protein from one strain and RNA from another) were true-to-type for the TMV nucleic acid ( Fraenkel-Conrat and Singer, 1957). Fraenkel-Conrat and Singer subsequently employed the mutagen nitrous acid ( Gierer and Mundry, 1958) to generate novel variants of TMV that were then compared in terms of their nucleic acid content, CP composition, and disease symptoms. Due to the labile nature of the TMV RNA, these were difficult experiments. As Singer recalled, she and Fraenkel-Conrat protected the RNA from cellular RNases by adding the clay bentonite, leading their colleague C.A. Knight to remark that he “wouldn't put that mud in his stuff.”

Singer asserted that her work with Fraenkel-Conrat represented the true beginning of chemistry applied to virology. However, one might well point out that this work drew on concurrent developments in bacteriology and bacterial genetics, beginning with research performed a decade earlier at the Rockefeller Institute, where Avery and his coworkers biochemically demonstrated the “transforming principle” of Streptococcus to be DNA.

With the elucidation of the complete CP sequence in 1960 ( Anderer et al., 1960 Tsugita et al., 1960) the collection of TMV mutants provided clues used to crack the genetic code. Only the startling development of cell-free translation systems the following year by H. Matthei and M. Nirenberg provided a less laborious means to decipher this code (reviewed in Kay, 1998), and even then TMV mutants were used to confirm the emerging codon dictionary.

The TMV mutants shed light on other biological questions as well. As Singer also noted, almost all the mutants attributed to the nitrous acid treatment were less “fit” than was wild-type TMV, an observation suggestive of later developments in the arenas of virus diversity and evolution.

Milton Zaitlin (Cornell University, Ithaca, NY) recalled how advances in molecular genetic techniques enabled researchers in the 1970s and 1980s to construct a detailed map of the TMV genome. Indeed, a significant clue to the genetic composition of TMV RNA came from studies in Zaitlin's laboratory showing that a low molecular–weight component termed sgRNA accumulated during viral infection ( Jackson et al., 1972). This sgRNA was soon implicated as the mRNA that directs CP production ( Hunter et al., 1976). By the mid-1970s, Zaitlin's group had correctly, albeit tentatively, placed the replicase-encoding gene at the 5′ end, the CP-encoding gene at the 3′ end, and a gene necessary for viral movement in the central portion of the genome ( Beachy et al., 1976 see Figure 1). Nishiguchi, Okada, and coworkers ( Nishiguchi et al., 1978 Ohno et al., 1983) then confirmed that the 30-kD MP was encoded by TMV using TMV strain L and a temperature sensitive variant, Ls-1. Several reverse genetics studies using infectious clones, first assembled in 1986 ( Dawson et al., 1986 Meshi et al., 1986), have confirmed the gene functions assigned during these earlier studies.

The initiation of TMV infection and disassembly of the TMV virion was reviewed by John G. Shaw (University of Kentucky, Lexington). Having entered its host cell, the TMV virion must remove its CP to enable viral replication. Shaw presented one model describing how this uncoating might take place bi-directionally, proceeding both from the 5′ and the 3′ ends of the TMV genomic RNA molecule. The 5′-to-3′ uncoating reaction may be cotranslational ( Wilson, 1984), which would result in disassembly by a ribosome-mediated mechanism and concomitant protection of the uncoated viral RNA from cellular nucleases. Shaw suggested that the 3′-to-5′ uncoating reaction might occur in a coreplicational manner, because viral replicase mutants that are defective in 3′-to-5′ disassembly can be uncoated in plant protoplasts by adding free viral RNA with an intact replicase gene (Wu and Shaw, 1996, 1997). These studies of TMV infection at the molecular level exemplify the highly efficient coordination of seemingly disparate events associated with virus replication.

Ken Buck (Imperial College of Science, Technology, and Medicine, London, UK) dissected the process of TMV replication, and he noted that although the viral proteins involved in TMV replication are well characterized, the involvement of host factors is poorly understood. On the basis of their physical association with the viral replicase proteins, Buck proposed two candidate host proteins that might be involved in TMV RNA synthesis: EF-1α, which colocalizes with the replicase complex, and a subunit of eIF-3, which copurifies with the replicase. In addition, Buck mentioned genetic approaches to dissect virus–host interactions that have led to the identification of the tom-1 and tom-2 mutants of Arabidopsis and the Tm-1 mutant of tomato, in which TMV replication is restricted.

Population genetic studies with tobamoviruses have also yielded surprising results. Although RNA viruses have the potential to vary more widely than DNA viruses ( Domingo and Holland, 1994), TMV provides a case of high genetic stability. Adrian J. Gibbs (Australian National University, Canberra) compared cDNA sequences of recent isolates of tobacco mild green mottle tobamovirus (TMGMV) and TMV with those derived from infected Nicotiana glauca specimens deposited in Australian herbaria since 1899. Gibbs reported that these analyses demonstrate that there has been no increase in the genetic diversity of TMGMV in Australia over the past 100 years. Moreover, the mutations observed in TMV seem to be deleterious because TMGMV has become the more dominant tobacco virus in N. glauca in that country ( Fraile et al., 1997).

More generally, tobamoviruses from places as far removed as California and Crete appear to be part of one large world population with very limited variation. This remarkably constrained variation suggests that, despite varying selective pressures, the viral genome remains generally immutable as a result of long-term host–virus interactions. In other words, there appears to be a restricted window of TMV sequence variability, outside of which the host plant's ability to recognize and repulse this pathogen is greatly enhanced. In this respect, TMV appears to be very different from other viruses, such as influenza and HIV, which characteristically exhibit high rates of nucleotide change. The restricted variation characterizing TMV worldwide likely aided early virologists, who were able to duplicate results from distant laboratories with relative ease.


Discussion

Early Stages of TMV Infection

The replication of many positive-strand RNA viruses occurs in close association with membranes (e.g., Wu et al. 1992 Molla et al. 1993 Strauss and Strauss 1994 Osman and Buck 1996). In this study, TMV vRNA was specifically localized with several types of cellular membranes. Early in infection, vRNA was associated with a perinuclear reticulated network and with strands of tubules and small vesicles. These structures closely resembled the cortical polygonal ER network of tubules and sheets with intervening lamellar segments connected to the nuclear envelope, previously described in plant cells (Allen and Brown 1988 Hepler et al. 1990 Staehelin 1997). Colocalization of vRNA with BiP, a resident protein of the ER (Denecke et al. 1995 Boston et al. 1996) and the disruption of the fluorescent structures by BFA (Fig. 8), support the hypothesis that vRNA is associated with ER. Our observations that viral replicase (Ishikawa et al. 1986) and the complementary minus-strand RNA (used as template for vRNA replication) were localized in these membranous structures strongly suggest that replication of TMV vRNA takes place in close association with the ER. Earlier reports described a relationship between TMV replication complexes and membranous extracts from infected cells (Watanabe and Okada 1986 Osman and Buck 1996).

Minus-strand RNA was also localized to structures (presumed to be ER) that surround the nucleus, with the exception of a discrete region. The absence of minus-strand RNA from this region could reflect compartmentalization of the perinuclear ER, or it may indicate that the ER is divided into subdomains with specific morphological or functional properties (Staehelin 1997). Ishikawa et al. 1986 suggested that the synthesis of plus and minus strands of vRNA requires different types of factors or molecular interactions, an observation that may have relevance to our studies.

vRNA and viral replicase were colocalized in small patches that are distributed throughout the cytoplasm. As previously indicated in poliovirus infection (Bienz et al. 1983), we suggest that patches that contain vRNA and replicase correspond to replication complexes in association with ER that compartmentalize the required components of replication to enhance virus production.

At early stages of infection, vRNA was associated with fluorescent filaments that resembled elements of the cytoskeleton (not shown). Based on the effects of oryzalin and cytochalasin D on the distribution of vRNA in the cytoplasm, we suggest that there is association of vRNA with the cytoskeleton at a very early stage of infection. In this scenario, vRNA exploits the cytoskeleton for transport from the cytoplasm to perinuclear positions. This hypothesis is based in part on the observation that treating protoplasts with oryzalin at the time of inoculation prevented the localization of vRNA to the perinuclear region. A recent report described a mechanism in newt lung epithelial cells by which the ER membranes attached to microtubules are transported toward the cell center through actomyosin-based retrograde flow of microtubules with ER attached as cargo (Waterman-Storer and Salmon 1998). Following this model, it is possible that vRNA-replicase complexes associated with ER are transported via microtubules to perinuclear positions using a similar mechanism.

Throughout the infection, vRNA was localized in different subcellular compartments. We suggest that this reflects movement of vRNA to different compartments, but cannot eliminate the possibility that compartmentalization represents the synthesis and subsequent degradation of vRNA rather than movement of vRNA from one compartment to another.

When protoplasts were infected with vRNA-ΔM, a mutant of TMV that does not produce MP, vRNA-ΔM was localized to vesicle-like structures around the nucleus and in small patches in the cytoplasm. These results indicate that at an early stage of infection, association with the ER is an intrinsic property of vRNA and/or the replicase and does not require MP. It is possible that vRNA contains sequences that target to the ER. Some cellular mRNAs are known to contain specific signals that direct them to the rough ER for translation (St. Johnston 1995).

Midstages of Infection: Virus Accumulation and Intracellular Transport

At midstages of infection, vRNA was localized in fluorescent, irregularly shaped bodies, some of which were vesicle-like in appearance. Furthermore, the replicase (Fig. 4) and MP (Fig. 5) colocalized with vRNA on these bodies. Since such structures were not observed in protoplasts infected with vRNA-ΔM, we conclude that MP is required for the formation and/or stabilization of the bodies. These structures may correspond to the previously described “viroplasms or amorphous inclusions” induced by TMV infection (Martelli and Russo 1977). Our observations support an earlier suggestion that replication complexes associated with rough ER function as mRNAs (Beachy and Zaitlin 1975). During its synthesis, MP may remain associated with vRNA, acting as an anchoring protein and trapping vRNA on ER-derived structures. Accordingly, the large accumulation of MP is coincident with dramatic morphological changes that take place on the ER (Reichel and Beachy 1998).

Over time, the cytoplasmic bodies become elongated structures, often associated with fluorescent filaments, directed toward the periphery of the cell. Immunostaining with antitubulin antibody and in situ hybridization reactions provide clear evidence of colocalization of vRNA with microtubules. Treatment of protoplasts at midstage of infection with oryzalin prevented the dispersion of the bodies to the periphery of the cell, suggesting that microtubules play a role in intracellular distribution of vRNA. Two different microtubule-based mechanisms, membrane sliding and tip attachment complexes, participate in the movement of ER from the cell center to the periphery of newt lung epithelial cells (Waterman-Storer and Salmon 1998). In TMV infection, the complexes that contain MP, vRNA, and replicase that are associated with ER may be transported towards the periphery of the cells using such mechanisms.

Treatment of infected protoplasts with cytochalasin D clearly altered the pattern of vRNA distribution and caused a delay in the appearance of the large bodies, suggesting a role of microfilaments in their formation and/or stabilization. However, depolymerization of microtubules can also lead to disruption of microfilaments (Panteris et al. 1992) it is therefore difficult to define the specific role of each cytoskeletal component in the intracellular spread of vRNA.

vRNA-ΔM was not associated with elements of the cytoskeleton unless the cells were treated at early stages of infection with cytochalasin D. Such treatment resulted in accumulation of vRNA-ΔM in fluorescent spots at the periphery of the cell, as well as on filaments that were similar in appearance to microtubules. These results suggest that vRNA was associated with microtubules when both MP and microfilaments were absent. The transport of mRNAs along cytoskeletal components has been described in a variety of biological systems, especially in relation to cell differentiation and development (Ferrandon et al. 1994 Kloc and Etkin 1995 Ainger et al. 1997 Broadus et al. 1998). In those cases, RNA transport mechanisms involved specific sequences in the 3′ untranslated region that are recognized by proteins that bind these sequences and mediate the interaction with the cytoskeleton (Oleynikov and Singer 1998). In our study, the change in the distribution pattern of vRNA that is induced by cytochalasin D was more evident in protoplasts infected with vRNA-ΔM vs. wt vRNA. These data suggest some influence of the MP in localization of vRNA in cytochalasin D–treated protoplasts. The implications of MP and microfilaments in the formation and anchoring of the cytoplasmic bodies are discussed below.

Late Stages of Infection: Virus Spread and/or Degradation?

Throughout infection, vRNA was localized around the nucleus, although at mid and late stages, vRNA was also dispersed throughout the cytoplasm and at the periphery of the cell. Accumulation of vRNA around the nucleus may facilitate association with host components that are required for virus infection. However, the accumulation of the much larger bodies that surround the nucleus may imply a different biological role in replication. Recently, a model was proposed by which misfolded proteins that escape the proteosome-mediated pathway of degradation can aggregate to form large structures, referred to as “aggresomes.” Aggresomes are transported on microtubules from peripheral sites to a ubiquitin-rich nuclear location at the microtubule-organizing center, where they are entangled with collapsed intermediate filaments (Johnston et al. 1998). Following this model, during TMV infection, the bodies that contain MP, vRNA, and replicase may be transported on microtubules in order to be degraded. In other recent studies, we described the ubiquitination of MP and the role of proteosomes in degradation of MP (C. Reichel and R.N. Beachy, manuscript submitted for publication).

At late stages of infection, vRNA was localized in structures that protrude from the surface of the cell. These protrusions were not disrupted by oryzalin or cytochalasin D. Furthermore, in other studies, we observed that the protrusions were stained with DiOC6(3) (3, 3′-dihexyloxacarbocyanine iodide), a vital fluorescent stain of ER (not shown). Other studies indicate that these ER-containing structures are not induced by virus infection per se, but may be stimulated by infection (P. Más and R.N. Beachy, manuscript in preparation). We propose that the protruding structures are related to desmotubules, the appressed ER that comprises the central component of plasmodesmata (Lucas et al. 1993). Alternatively, they could be a consequence of cell damage at late stages of infection, which results in the extrusion of ER through the plasma membrane. Interestingly, vRNA-ΔM was not associated with the protrusions, indicating a role of the MP in localization of vRNA with these structures. Together, these results may imply that at least two different types of ER are involved in TMV infection. One type of ER is involved in vRNA replication and does not require the presence of the MP. A second type corresponds to the filamentous protrusions that may be involved in the intercellular spread of the virus and requires a functional MP.

Model of TMV Infection

The data presented here and in previous publications are consistent with a model of TMV infection (Fig. 11) in which the replication of vRNA takes place in close association with membranes of the ER (Fig. 11, Fig. 1). In this model, cytoskeletal elements are involved in targeting vRNA/replicase complexes to the perinuclear ER, perhaps via a retrograde flow of microtubules with ER attached as cargo. The ER-associated nascent vRNAs in replication complexes function as mRNAs for the synthesis of MP (Fig. 11, Fig. 2). The MP remains associated with vRNA in the complex, resulting in the formation of large ER-derived structures (Fig. 11, Fig. 3). At this point, the distribution of vRNA would be determined by a balance between the formation and anchoring of the large structures and their spread towards the periphery of the cell. Our results are consistent with a model in which MP and microfilaments participate in the formation and anchoring of the ER-derived structures (Fig. 11, Fig. 3), while microtubules are involved in the transport to their final destinations i.e., to the periphery for intercellular spread, or toward the nucleus for degradation (Fig. 11, Fig. 4).

Several types of experimental evidence support this model. First, there is a dramatic effect on distribution of vRNA-ΔM in cytochalasin D–treated protoplasts. Not only were bodies not found in these protoplasts, in contrast to wt vRNA, but vRNA-ΔM was located on or near the cell periphery but not in the protrusions from the plasma membrane. Since in nontreated protoplasts vRNA-ΔM was not localized at the periphery in early stages of infection, the intracellular spread that normally occurs later in infection was apparently accelerated in the absence of microfilaments. Second, the clear association of vRNA-ΔM with microtubules would explain the role of microtubules in the intracellular spread of the virus towards the periphery of the cell. Third, at late stages of infection, the close relationship between the ER and microtubules explains the association of vRNA in the presence of the MP to a precursor of the plasmodesmata (Fig. 11, Fig. 5) that would result in the cell-to-cell spread of the virus in leaf tissues.


Conclusion

A simple, cost effective, and efficient way to infect plants with TMV vectors via agroinfection was identified. Recombinant protein expression levels of approximately 600 to 1200 micrograms per gram of A. tumefaciens infiltrated plant tissue were obtained. Recombinant protein can be recovered from both locally inoculated leaves or from systemically infected plant tissue. These improvements should enable researchers with little or no experience with plant virus vectors to easily utilize TMV expression vectors in their research. The vector improvements described here will be especially useful in high-throughput research projects.


Discussion

Plants, as sessile organisms, need to respond to biotic or abiotic stresses by rapidly readjusting the abundance levels of specific proteins with a critical role to stresses. The orchestrated up- and downregulation of specific host factors in virus–plant interaction can result in a defense reaction against the virus. Alternatively, one could hypothesize that such alterations of the host factors will benefit the invading virus in a compatible interaction. The literature reports that protein changes are associated with a putative defensive role against TMV infection in tobacco, while other protein changes are associated with a successful TMV infection in tobacco-causing disease. Our proteomics analysis identified 661 proteins. Tobacco has an incomplete protein database to date (6,793 accessions) and a low annotated rate that could explain the low identification rate. The use of gel-free proteomics for protein identification requires a well-annotated database of the host species under study to be able to identify numerous proteins.

Despite the relatively low number of identified proteins, we found a significant correlation to the different treatments. Thirty-six (36) proteins were found to be differentially accumulated in the tobacco–TMV compatible interaction as early as 15 mpi. The protein changes that could contribute to resistance to TMV involve defense-related proteins such as S-adenosylmethionine synthase, cysteine proteinase inhibitor, glutathione S-transferase, malate dehydrogenase, Snakin-1, osmotin-like protein, and RNA-binding glycine-rich protein (upregulated upon TMV infection), as well as putative susceptibility factors such as phosphoglycerate kinases and OBERON-like protein (downregulated upon TMV infection) (Table 1).

S-adenosylmethionine synthase or SAMS (A0A0F7R532) catalyzes the formation of S-methylmethionine (SMM), which is necessary for the production of several osmoprotectant proteins (Espartero et al., 1994). Elevated levels of SMM could contribute to a response against plant stress, as it is a direct precursor of the osmoprotectant sulfoniopropionate family of proteins involved in both biotic and abiotic defense mechanisms. SMM also influences the biosynthesis of regulatory and defense compounds such as polyamines and ethylene (Tassoni et al., 2008). Cysteine proteinase inhibitor (A0A075F933) putatively plays a role in plant defense against viral infections (Shih et al., 1987 Prins et al., 2008). Gutierrez-Campos et al. (1999) expressed plant cysteine protease inhibitors in transgenic tobacco conferring resistance against potyviruses. Glutathione S-transferase or GST (Q4LB98) is involved in the neutralization of toxic compounds and reactive oxygen species formed during virus infection (Zechmann et al., 2005). In the case of Bamboo mosaic virus, plant GST binds to the viral RNA delivering glutathione to the viral replication complex (Chen et al., 2013). Glutathione is a key regulator of redox signaling and buffering and plays an important role in plant defense through the activation of defense-related genes (Foyer and Noctor, 2009). Malate dehydrogenase (A0A075F1V0) reversibly catalyzes the conversion of oxaloacetate to malate in both mitochondria and the cytoplasm, leading to the production of secondary metabolites (Tomita et al., 2005), such as alkaloids, flavonoids, and terpenoids, which are made in the reaction of mechanical damage or infection (Beggs and Wellman, 1994). The antimicrobial peptide Snakin-1 or SN1 (A0A0R4WFT2) has been associated with enhanced resistance against bacteria, fungi, and viruses (Almasia et al., 2008 Rong et al., 2013 He et al., 2017). The SN1 gene of soybean was found to enhance virus resistance in Arabidopsis and soybean probably by altering the expression of signal transduction and defense response genes (He et al., 2017). Osmotin-like protein (A0A075EZS9) belongs to the pathogen-related protein-5 (PR-5) family of proteins with a putative defensive role against several pathogens, besides its role as a osmoregulator. A higher abundance of osmotin-like proteins in TMV-infected leaf tissues has been reported (Broekaert et al., 1997). The glycine-rich RNA-binding protein or GRP (J7G1D7) participates in host–pathogen interactions, having a role in nucleic acid binding, hypersensitive response, and salicylic acid biosynthesis (Naqvi et al., 1998). Overexpression of the A. thaliana glycine-rich RNA-binding protein 7 (AtGRP7) conferred resistance against TMV (Lee et al., 2012). Furthermore, ADP ribosylation of the RNA-binding proteins attenuated host immunity by affecting RNA metabolism and the plant transcriptome related to defense (Fu et al., 2007).

Phosphoglycerate kinases or PGKs (Q42961, Q42962) were shown to promote the replication of several positive-strand RNA viruses (Cheng et al., 2013 Chen et al., 2017 Prasanth et al., 2017). This may be achieved either by their ATP-generating activity that facilitates the establishment of viral replication complexes (VRCs) (Prasanth et al., 2017) or by their viral RNA-binding capacity that helps the transport of viral RNA inside chloroplasts for replication (Cheng et al., 2013). Interestingly, a naturally occurring mutant of PGK in A. thaliana exhibits resistance to a potyvirus, suggesting that PGKs may function as host factors that increase susceptibility to virus infection (Ouibrahim et al., 2014). OBERON-like protein (Q84N38) was shown to promote systemic spreading of the potyvirus Turnip mosaic virus (TuMV) via interaction with the Vpg viral protein. Downregulation of OBERON-like protein decreases TuMV infection (Dunoyer et al., 2004).

All the above-described proteins, identified by proteomics analysis, are involved in the efforts of the host to defend itself against TMV at a very early infection stage, however, without success since plants are systemically infected by the virus. The protein changes correlated with a successful TMV infection involve the translation-associated poly-A binding protein (upregulated upon TMV infection), as well as chloroplast-associated factors such as carbonic anhydrases, chloroplast photosystem bO4 protein, and RuBisCO activase 2 (downregulated upon TMV infection) (Table 1 and Figure 2).

Poly-A-binding protein or PABP (I2FJN7) may act as a susceptibility host factor by promoting viral RNA translation (Iwakawa et al., 2012). TMV 3′ UTR contains several unique sequences designated as CAP-independent translation enhancer element (CITE) and A-rich sequences (ARS) which mediate viral RNA translation.

Carbonic anhydrase or CA enzymes (EC 4.2.1.1) (A0A077DCK9, A4D0J8, A4D0J9, P27141) have been identified as salicylic acid-binding proteins with an antioxidant role and generally constitute part of the defense mechanism in C3 plants upon attack by various pathogens, including viruses (Slaymaker et al., 2002 Restrepo et al., 2005). PsbO group protein D1 (I0B7J4) was shown to exhibit significantly lower abundance in TMV-sensitive tobacco cultivars upon TMV infection compared with the TMV-tolerant ones, indicating that high D1 levels are tightly correlated with antiviral resistance (Wang et al., 2016). In addition, silencing of PsbO in N. benthamiana resulted in increased TMV accumulation (Abbink et al., 2002). PsbO proteins are involved in the control of the photosystem II (PSII) affinity for manganese (Mn 2+ ), thus participating in the stabilization of the oxygen-evolving complex (Popelkova et al., 2008). An intact and operational PSII complex is crucial for resistance to TMV infection (Wang et al., 2016). Interestingly, TMV p126 helicase protein interacts with a PsbO protein in a yeast two-hybrid system suggesting that p126 may interrupt PsbO’s normal localization and/or function (Abbink et al., 2002 Wang L. Y. et al., 2012). RuBisCO activases or RCAs (Q40565, V9INR4), being chaperone-like proteins, are required for optimizing photosynthesis and were found to colocalize with TMV replicase inside VRCs upon TMV infection. Downregulation of RCA substantially increases the infection of TMV (Bhat et al., 2013). Chloroplasts are energy generators, stress sensors, and defense signal producers and thus constitute major targets of invading viruses to establish successful infections (Li et al., 2016 Bhattacharyya and Chakraborty, 2018). Our findings showing the decrease in CA, PsbO, and RCA protein levels at 15 mpi could represent the beginning of a rapid alteration of the chloroplast caused by TMV (Table 1 and Figure 2). Typical TMV disease symptoms on tobacco leaves such as mosaic are correlated with abnormal chloroplast structure and distortion of the photosynthetic machinery (Lehto et al., 2003).

RNAi is considered a very effective antiviral mechanism (Padmanabhan et al., 2009 Wang M. B. et al., 2012). In addition, it was proposed recently that dsRNA could induce a pattern-triggered immune (PTI) signaling pathway in plants providing antiviral defense (Niehl et al., 2016 and references therein). No studies have been reported related to the production of vsiRNAs from the TMV genome as early as 15 mpi. Tobacco RNAi against TMV is operational at this time point in the tobacco–TMV interaction, since vsiRNAs from sense and antisense orientation of the TMV genome are produced (Figure 4 and Supplementary Table 5). However, even this defense reaction is not sufficient to confer resistance since all tobacco plants got infected. To boost the host RNAi machinery, we employed the topical application of dsRNAs, as inducers of the RNA-based vaccination (Voloudakis et al., 2018). In the present work, we used small RNA NGS to show that the exogenously applied dsRNAp126 on tobacco plants is efficiently processed as early as 15 mpa, producing siRNAs derived from the entire region of the dsRNA molecule used (Figure 3 and Supplementary Table 4), and this is to our knowledge reported for the first time. Since the siRNA profiles are similar in TMV and dsRNAp126 treatments (Supplementary Figure 3), we assume that DCLs play a key role in dicing the exogenously applied dsRNA. The observed heterogeneity in siRNA production (hot spots) (Figure 3) could be possibly attributed to the secondary structures of the dsRNA molecules that may restrict the accessibility of the DCL proteins to their targets. TMV via its p126 protein, which possesses RNA silencing suppressor activity, could interfere with the dicing capacity of the host DCL proteins, affecting as a result the exogenously applied dsRNA processing. However, this does not seem to happen in our experimental system. In particular, the simultaneous topical application of dsRNAp126 along with TMV produced the same pattern of siRNA molecules in the region 426𠄱,091 nt (region of dsRNA) (Figures 3, 5 and Supplementary Figure 2), suggesting that the presence of the virus does not alter the dicing pattern of the dsRNA applied. The siRNAs produced, at 15 mpi, in the region of dsRNAp126 (426𠄱,091 nt) is in great excess relative to the vsiRNAs derived from the rest of the viral genome (Figure 5 and Supplementary Table 6). This high abundance of dsRNA-derived siRNAs is a very important parameter for RNA-based vaccination since RNAi efficacy was previously shown to be dose dependent (Tenllado and D໚z-Ruíz, 2001). The efficacy of dsRNAp126 application against TMV infection can be illustrated by the finding that it significantly reduces the accumulation of TMV, as evidenced by RT-PCR analysis (Figure 6).

1-Aminocyclopropane-1-carboxylate oxidase 2 isoform A (ACO2) (E5LCN0), cysteine synthase (CyS) (Q3LAG5, S6A7M4), and isoforms of calmodulin (CaM) (Q76ME6, Q76MF3) compose a small group of proteins that were significantly more abundant in the dsRNAp126 + TMV treatment when compared with the water and TMV treatments. We hypothesize that these proteins are induced by dsRNA itself. No bibliographic reports exist involving ACO2 and CyS in plant virus infection. However, a calmodulin-like protein (rgd-CaM) suppresses the Cucumber mosaic virus-2b (CMV-2b), the viral silencing suppressor, by physical binding to CMV-2b’s dsRNA-binding domain (Nakahara et al., 2012). It would be important to further investigate the accumulation of these proteins to confirm their involvement in plant RNAi pathway and resistance to virus infection.


Structure–function analysis of n

The presence of TIR, NB and LRR structural motifs in the N gene implies that it is involved in protein complexes that recognize the TMV ligand and trigger signal transduction leading to the defence response. Deletion- and site-directed mutagenesis of key amino acid residues in these three domains indicated that all three domains are required for proper N function ( Dinesh-Kumar et al., 2000 ). Most in-frame deletion mutations in the N gene abolished TMV resistance and were recessive to wild-type N-gene function. The TIR deletion mutants showed a dominant negative phenotype in a wild-type NN background.

The N-TIR domain was further characterized by targeting nine amino acids in this domain that are conserved in plant R gene products, as well as in those of Drosophila and human Toll, and human IL-1R ( Rock et al., 1998 ). These amino acids are essential for the activation of signalling events in the Toll and IL-1R pathways. Some of the amino acid substitutions within the TIR domain caused a complete loss of N function ( Dinesh-Kumar et al., 2000 ). For example, though the substitution mutation D46Y had no effect on N function, D46H resulted in non-functional N. Similarly, conservative or non-conservative substitutions at positions Y12, Q67, W82, I138, W141 and R142 led to partial loss of function phenotype. The substitution Y12F showed no effect while the timing and appearance of HR was different in plants bearing the Y12S mutation. Irrespective of the timing or appearance of HR, all of these mutants developed the SHR phenotype. Some of the loss-of-function and partial loss-of-function alleles acted in a dominant negative fashion and interfered with the wild-type N function. One critical outcome of these studies was that mutations that affect Drosophila Toll or human IL-1R signalling were also found to affect the N-mediated resistance response to TMV infection. However, the exact role of the N-TIR domain in TMV resistance signalling is still unknown.

The NB domain of the N gene and other R genes share amino acid sequence homology with regions of cell death genes, CED4 in C. elegans and Apaf1, FLASH, CARD4 and Nod1 from humans ( van der Biezen and Jones, 1998 Arvind et al., 1999 ). The NB domain is also found in the RAS group, ATPases, elongation factors and G-Protein families of proteins ( Saraste et al., 1990 ). These serve as molecular switches in various cellular functions, such as growth and differentiation. The role of the NB region in the TMV resistance pathway was evaluated by introducing 20 point mutations in seven conserved amino acids across three subdomains of NB: P-loop (kinase1), kinase2a, and kinase3 ( Dinesh-Kumar et al., 2000 ). Any substitution in the invariant G216 or K222 of P-loop of N led to loss of resistance. It is worth noting that mutations in the G12 and G13 amino acids in RAS play an important role in oncogenic transformation and these mutants display reduced GTPase activity. Corresponding mutations in the N-gene, at residues G218 and G219, led to partial or complete loss-of-function phenotypes. Interestingly, some of these mutants are dominant over the wild-type N allele. It is known that the hydroxyl group of serine or threonine in the P-loop is involved in the binding of Mg 2+ associated with bound nucleotides ( Pai et al., 1990 ). The T223S substitution at the putative Mg 2+ binding site of the N gene led to a partial loss-of-function phenotype. The timing of HR appearance and size of HR lesions was normal, but TMV spread throughout the plant and resulted in the death of the entire plant within 5–7 days. Interestingly, replacing T223 with aspargine or alanine resulted in loss of N function, leading to mosaic symptoms. However, these alleles are dominant over the N gene-induced HR and resistance responses. Despite the requirement for the NB domain for disease resistance function, there is no biochemical evidence to support direct binding of nucleotides to the NB domain of R proteins.


RESULTS

TMV-RNA induced autophagy in HeLa cells

Transfection of TMV-RNA into HeLa cells

Because there is no receptor for TMV on human cellular membranes, TMV was not able to enter HeLa cells when TMV was co-cultured with HeLa cells, CP was not detected in HeLa cells. To import intact TMV particles or TMV-RNA into HeLa cells, we used two methodologies: electroporation or liposome transfection, respectively. Following the electroporation procedure [27], intact TMV particles were transformed into HeLa cells by electric shock with different voltages (0, 140, 280 and 360 V). Western blotting results showed that there was a little more CP expression in HeLa cells treated with 280 V than with the other voltages, but not high enough in CP expression (Figure 1A-a). This result suggests that some cells may have been fatally damaged by the strong electric currents. Thus we concluded that electroporation was not the proper method for TMV transfection. Recent reports have demonstrated that the liposome transfection method is more effective and less toxic to cells than other transfection methods [28]. Thus we used this method for TMV-RNA transfection. HeLa cells were incubated with a mixture of TMV-RNA and liposomes at ratios of 1:1 and 1:2 for 6 h. Western blot analysis showed that CP was produced more abundantly at the ratio of 1:1 than 1:2 (Figure 1A-b), indicating that TMV-RNA can be effectively transfected into HeLa cells by using an equal proportion of liposomes. Moreover, the morphology of the cells did not change during the process of incubation. Thus we found an effective way to import TMV-RNA into HeLa cells, and this technique was applied exclusively in the following experiments.

TMV-RNA induces autophagy in HeLa cells

(A) Transfection of TMV or TMV-RNA into HeLa cells. (a) TMV was transfected into HeLa cells by electroporation at voltages of 0, 140, 280 and 350. Western blotting was performed for TMV CP using lysates from HeLa cells transfected with TMV for 24 h. (b) TMV-RNA (T-RNA) was transfected into HeLa cells using liposomes at ratios of 1:1 and 1:2 (T-RNA/Lipo) HeLa cells treated with liposomes only (Lipo) were used as the negative control and those treated with wild-type TMV were used as the positive control. Western blotting was performed for CP using lysates from TMV or HeLa cells transfected for 24 h. More CP was detected at ratios of 1:1 than 1:2. Thus liposome transfection of TMV-RNA was more effective and less toxic to HeLa cells than electroporation. β-Actin was used as a loading control. (C) Transmission electron micrographs of the ultrastructure of HeLa cells with or without TMV-RNA at different H.P.T. times. Autophagic vacuoles and acidic vesicular organelles were observed in the cytoplasm (arrowheads), and the vacuoles increased with increasing transfection time. Con, Control, HeLa cells without TMV-RNA transfection Lipo, HeLa cells treated with liposomes only. Scale bar: 2 μm. (D) The average number of vacuoles per cell was analysed in at least 100 randomly chosen TEM fields. Scale bar: 1 μm. *P<0.001. (B) Western blotting was performed for LC3 using lysates from HeLa cells transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h. β-Actin was used as a loading control. (E) An immunoblot-based LC3 flux assay by flow cytometry was used to monitor alterations in TMV-RNA-mediated autophagic flux. Analysis of LC3 in HeLa cells revealed that the percentage of cells with autophagosomes increased markedly when they were transfected with PBS (Con) or TMV-RNA for 24 or 48 h, whereas fewer autophagosomes were observed in cells treated with 3-MA (an autophagy inhibitor, 5 mM). *P<0.001. All results are from five independent experiments.

(A) Transfection of TMV or TMV-RNA into HeLa cells. (a) TMV was transfected into HeLa cells by electroporation at voltages of 0, 140, 280 and 350. Western blotting was performed for TMV CP using lysates from HeLa cells transfected with TMV for 24 h. (b) TMV-RNA (T-RNA) was transfected into HeLa cells using liposomes at ratios of 1:1 and 1:2 (T-RNA/Lipo) HeLa cells treated with liposomes only (Lipo) were used as the negative control and those treated with wild-type TMV were used as the positive control. Western blotting was performed for CP using lysates from TMV or HeLa cells transfected for 24 h. More CP was detected at ratios of 1:1 than 1:2. Thus liposome transfection of TMV-RNA was more effective and less toxic to HeLa cells than electroporation. β-Actin was used as a loading control. (C) Transmission electron micrographs of the ultrastructure of HeLa cells with or without TMV-RNA at different H.P.T. times. Autophagic vacuoles and acidic vesicular organelles were observed in the cytoplasm (arrowheads), and the vacuoles increased with increasing transfection time. Con, Control, HeLa cells without TMV-RNA transfection Lipo, HeLa cells treated with liposomes only. Scale bar: 2 μm. (D) The average number of vacuoles per cell was analysed in at least 100 randomly chosen TEM fields. Scale bar: 1 μm. *P<0.001. (B) Western blotting was performed for LC3 using lysates from HeLa cells transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h. β-Actin was used as a loading control. (E) An immunoblot-based LC3 flux assay by flow cytometry was used to monitor alterations in TMV-RNA-mediated autophagic flux. Analysis of LC3 in HeLa cells revealed that the percentage of cells with autophagosomes increased markedly when they were transfected with PBS (Con) or TMV-RNA for 24 or 48 h, whereas fewer autophagosomes were observed in cells treated with 3-MA (an autophagy inhibitor, 5 mM). *P<0.001. All results are from five independent experiments.

TMV-RNA induced autophagy in HeLa cells

After we established a reliable transfection system, we monitored the morphological changes in HeLa cells after TMV-RNA transfection by light microscopy. At 24 h post-transfection, we found that some vacuoles were produced in the cytoplasm of the HeLa cells. After 72 h, the proportion of vacuoles was more than 50% per microscopic field of vision. To determine whether autophagy was triggered after transfection, the ultrastructure of HeLa cells transfected with TMV-RNA for 6, 24, 48 and 72 h was analysed by TEM. Numerous vacuoles and some membrane-bound vacuoles characteristic of autophagosomes (arrowheads) were observed in the cytoplasm of the transfected cells (Figure 1C, T-6 h, T-24 h). Moreover, typical mature autophagosomes with double-membrane vacuolar structures containing visible cytoplasmic contents increased over time (Figure 1C, T-48 h, T-72 h Figure 1D). Vacuoles were only rarely found in cells not transfected with TMV-RNA (Figure 1C, Con), and only a few vacuoles were observed in cells incubated with liposomes only (Figure 1C, Lipo). The latter result suggests that the quantity of liposomes administered was less toxic to cells.

As described above, LC3 is used as a specific marker for the autophagosomal compartment, and the induction of autophagy leads to the processing of LC3-I into LC3-II. To detect the conversion of LC3-I into LC3-II, cells transfected with TMV-RNA for 24, 48 and 72 h were analysed by Western blotting. We found that the conversion of LC3-I into LC3-II was augmented if TMV-RNA treatment was extended to 72 h (Figure 1B). To monitor alterations in autophagic flux mediated by TMV-RNA, an immunoblot-based LC3 flux assay using flow cytometry was performed to quantify the number of autophagosomes. As shown in Figure 1(E), cells with autophagosomes detected using an FITC-conjugated anti-LC3 antibody were increased in HeLa cells following TMV-RNA transfection, whereas the expression of LC3-II was almost undetectable in the untransfected cells. Moreover, 3-MA, a common inhibitor of autophagy [29], was used to block autophagy in HeLa cells. In TMV-RNA-transfected cells treated with 3-MA (5 mM, Sigma) for 24 h, autophagy was markedly inhibited. Altogether, these observations suggest that TMV-RNA induces autophagy in HeLa cells.

TMV-RNA is the key factor for triggering autophagy in HeLa cells

Activation of autophagy genes is a critical step in autophagosome formation [17]. To examine whether the key autophagy gene (Beclin1) [21] was synergistically activated during TMV-RNA-induced autophagy, real-time PCR and Western blotting were performed to monitor the nucleic acid and protein levels, respectively, of Beclin1.

Beclin1 mRNA increased markedly when the transfection time was extended to 72 h (Figure 2A). Simultaneously, Beclin1 protein markedly accumulated within 24 h in TMV-RNA-treated HeLa cells, and the accumulation of protein was much more pronounced when the transfection time was extended to 72 h (Figure 2B). Neither Beclin1 nucleic acid nor Beclin1 protein was found in HeLa cells not transfected with TMV-RNA. To verify that the activation of Beclin1 in HeLa cells was indeed associated with TMV-RNA-induced autophagy, a human breast-cancer cell line in which Beclin1 is monoallelically deleted and thus expressed at reduced levels (MCF7 cells) [30] was transfected with TMV-RNA using the same protocol. Additionally, to interfere with the expression of the Beclin1 gene in cells we also transfected a Beclin1 siRNA (small interfering RNA) vector into HeLa cells. The Beclin1 siRNA vector was transfected into HeLa cells 24 h before the transfection of TMV-RNA. Analysis of the expression of Beclin1 and LC3 in MCF7 cells or HeLa cells by Western blotting showed that no Beclin1 was detected in MCF7 cells with or without TMV-RNA transfection, and the expression level of LC3 in MCF7 cells did not change after transfection (Figure 2C). Moreover, the expression of both LC3 and Beclin1 in HeLa cells transfected with both the Beclin1 siRNA vector and TMV-RNA was reduced compared with cells transfected with TMV-RNA only (Figures 2B and 2C). Taken together, the invasion of TMV-RNA directly results in autophagy, and Beclin1 is the key autophagy gene in TMV-RNA-transfected HeLa cells.

TMV-RNA is the key factor that activates Beclin1 in HeLa cells

(A) Real-time PCR analysis of Beclin1 mRNA from HeLa cells transfected with PBS (Con) or TMV-RNA for 6, 24, 48 or 72 h. Beclin1 mRNA increased markedly. *P<0.001. (B) Western-blot analysis of Beclin1 using lysates from HeLa cells transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h. Beclin1 was activated and increased as the transfection time was extended. (C) Western-blot analysis of Beclin1 and LC3 using lysates from HeLa cells transfected with TMV-RNA for 24, 48 or 72 h in the presence of a Beclin1 siRNA vector. MCF7 cells were also transfected with PBS (Con) or TMV-RNA for 24 h. Neither the expression of Beclin1 nor the conversion of LC3-I into LC3-II differed significantly between MCF7 cells and HeLa cells. β-Actin was used as a loading control.

(A) Real-time PCR analysis of Beclin1 mRNA from HeLa cells transfected with PBS (Con) or TMV-RNA for 6, 24, 48 or 72 h. Beclin1 mRNA increased markedly. *P<0.001. (B) Western-blot analysis of Beclin1 using lysates from HeLa cells transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h. Beclin1 was activated and increased as the transfection time was extended. (C) Western-blot analysis of Beclin1 and LC3 using lysates from HeLa cells transfected with TMV-RNA for 24, 48 or 72 h in the presence of a Beclin1 siRNA vector. MCF7 cells were also transfected with PBS (Con) or TMV-RNA for 24 h. Neither the expression of Beclin1 nor the conversion of LC3-I into LC3-II differed significantly between MCF7 cells and HeLa cells. β-Actin was used as a loading control.

To obtain further direct morphological evidence of the links between TMV proteins and the intracellular autophagic vacuoles, immunoelectron microscopy was performed. Immunogold particles identifying TMV proteins mostly presented on the membranes of autophagosomes 24 h after transfection (Figure 3A-b) when the time was extended to 48 h, the immunogold particles markedly accumulated on or around the membranes of autophagosomes (Figure 3A-c). Neither immunogold particles nor autophagosomes were found in untransfected cells (Figure 3A-a).

Electron micrographs showing TMV protein on and around autophagosomal membranes

(A) Immunoelectron microscopy of HeLa cells transfected with PBS (Con, a) or TMV-RNA for 24 (T-24 h, b) or 48 h (T-48 h, c). (b) TP (TMV-protein) immunogold particles were mostly present on autophagosomal membranes (A, autophagosome). (c) TP immunogold particles (TP, arrowheads) accumulated on and around autophagosomal membranes (A, autophagosome). Scale bars: 0.5 μm, 100 nm (as indicated in the enlargements). (B) Ultrastructure of HeLa cells by EM. Suspected TMV virions (b, T, arrowheads), whose structure is similar to TMV virions in tobacco leaves (a, Tobacco, T), aligned in parallel form and accumulated around cytosolic vacuoles. Scale bars: 1 μm, 200 nm (as indicated in the enlargements).

(A) Immunoelectron microscopy of HeLa cells transfected with PBS (Con, a) or TMV-RNA for 24 (T-24 h, b) or 48 h (T-48 h, c). (b) TP (TMV-protein) immunogold particles were mostly present on autophagosomal membranes (A, autophagosome). (c) TP immunogold particles (TP, arrowheads) accumulated on and around autophagosomal membranes (A, autophagosome). Scale bars: 0.5 μm, 100 nm (as indicated in the enlargements). (B) Ultrastructure of HeLa cells by EM. Suspected TMV virions (b, T, arrowheads), whose structure is similar to TMV virions in tobacco leaves (a, Tobacco, T), aligned in parallel form and accumulated around cytosolic vacuoles. Scale bars: 1 μm, 200 nm (as indicated in the enlargements).

As the first study to investigate the invasion of TMV in human cells, we hoped to find intact TMV particles in HeLa cells if TMV had the ability to escape from immune defence and avoid being digested by the lysosome. Indeed, we observed suspected TMV virions in HeLa cells by TEM. As shown in Figure 3(B-b), the suspected TMV virions aligned in a parallel form and accumulated around vacuoles. The virions resembled bundles of threads that were inlaid in the cellular cytoplasm. The observed features were similar to the TMV virions observed in tobacco leaves [31] (Figure 3B-a), so we deduced that TMV-RNA may replicate in HeLa cells.

The potential mechanism of TMV-RNA-induced autophagy in HeLa cells

Based on the above surprising observations, we performed further experiments to support our hypothesis. As mentioned above, the genomic structure of TMV-RNA functions like that of mRNA in that it can be translated into proteins directly. TMV can utilize the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication and particle assembly. If the translation of TMV-RNA occurred in the ER, the accumulation of TMV protein would inevitably lead to cellular stress responses, such as ERS, which may promote autophagy in HeLa cells. To test this assumption, three approaches were taken.

TMV-RNA is translated in the ER membrane of HeLa cells

In eukaryotic cells, mRNAs are partitioned between the cytosolic and ER compartments. As described in current models, this ubiquitous mRNA partitioning process is driven by a co-translational cycle of ribosome binding and release into the ER. In brief, all mRNAs are translated in the cytosol. Early in synthesis, those mRNA/RNCs (ribosome/nascent polypeptide chain complexes) engaged in the synthesis of secretory/membrane proteins are trafficked to the ER because of the presence of an SRP (signal recognition particle) [32]. Therefore we sought to determine whether TMV-RNA was driven by a co-translational cycle of ribosome binding and translation of proteins in the ER and to confirm the location of TMV-RNA.

After the transfection of TMV-RNA for 24, 48 and 72 h, cell lysates were harvested and used for the detection of proteins by Western blotting. The results indicate that TMV-RNA was translated into CP CP markedly increased within 24 h in TMV-RNA-treated HeLa cells, and the accumulation of the protein increased further when the transfection time was extended to 72 h, whereas it was undetectable in HeLa cells not transfected with TMV-RNA (Figure 4A). Considering the high-expression level of CP in HeLa cells, we deduced that TMV might escape from immune defence. To determine whether TMV-RNA was translated in the ER, we isolated the ER from HeLa cells transfected with TMV-RNA for 24, 48 and 72 h. Analysis of CP in the ER fraction by Western blotting showed that the expression level of CP markedly increased when the time was extended to 72 h (Figure 4A). To further confirm whether CP was located in the ER and to observe the location of TMV-RNA, we performed immunofluorescence (IF) and FISH techniques [25] in succession. TMV-positive RNA and proteins were identified in situ by confocal microscopy in cells fixed by a protocol intended to retain native cell size and shape. The TMV-positive RNA was visualized by FISH with a strand-specific probe that was labelled with a Cy3 fluorochrome (red). CP was detected using a rabbit anti-CP antibody and a corresponding secondary antibody (Alexa Fluor® 488 anti-rabbit antibody) (green), and the ER was stained with ER tracker (blue, Invitrogen). As shown in Figure 4(B), blue, red and green fluorescence indicated the locations of the ER, TMV-positive RNA and CP, respectively, in HeLa cells transfected with TMV-RNA for 48 h [H.P.T. (hours post-transfection) 48 h]. We observed that CP (green) indeed accumulated in the ER as small, irregularly sized granules, whereas TMV-RNA (red) rarely appeared in the ER but was enriched in the nucleolus. Neither CP nor TMV-positive RNA was found in cells not transfected with TMV-RNA (Figure 4C-c).

TMV-RNA is translated into CP and further augments the ER of HeLa cells

(A) Western blot of CP using lysates from HeLa cells [transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h] or the ER fraction of HeLa cells (transfected with TMV-RNA for 24, 48 or 72 h). TMV-RNA was translated into CP in HeLa cells and accumulated in the ER as the transfection time was extended to 72 h. (B) Combined IF and RNA-FISH for detecting CP and TMV-positive RNA. HeLa cells were transfected with PBS (Con) or TMV-RNA for 48 h. The cells were fixed and permeabilized to detect CP using a rabbit anti-CP antibody and a corresponding secondary antibody (Alexa Fluor® 488 anti-rabbit antibody) (green). The same cells were then treated for FISH analysis. Cells were hybridized with a probe labelled with a Cy3 fluorochrome (red) to detect TMV-positive RNA (B-c, T-RNA). The ER was stained with ER tracker (blue, B-d C-b, ER). The cells were then observed by confocal microscopy. (a) HeLa cells (B, C), DIC (differential interference contrast) CP (green, B-b) remained largely at the ER (B-e: merged b and d) TMV-positive RNA (red, B-c, T-RNA,) was rarely located on the ER (B-f: merged c and d), but it appeared in the nucleus. (C) Neither CP nor TMV-positive RNA was found in cells transfected with PBS (C-c) (scale bar: 10 μm).

(A) Western blot of CP using lysates from HeLa cells [transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h] or the ER fraction of HeLa cells (transfected with TMV-RNA for 24, 48 or 72 h). TMV-RNA was translated into CP in HeLa cells and accumulated in the ER as the transfection time was extended to 72 h. (B) Combined IF and RNA-FISH for detecting CP and TMV-positive RNA. HeLa cells were transfected with PBS (Con) or TMV-RNA for 48 h. The cells were fixed and permeabilized to detect CP using a rabbit anti-CP antibody and a corresponding secondary antibody (Alexa Fluor® 488 anti-rabbit antibody) (green). The same cells were then treated for FISH analysis. Cells were hybridized with a probe labelled with a Cy3 fluorochrome (red) to detect TMV-positive RNA (B-c, T-RNA). The ER was stained with ER tracker (blue, B-d C-b, ER). The cells were then observed by confocal microscopy. (a) HeLa cells (B, C), DIC (differential interference contrast) CP (green, B-b) remained largely at the ER (B-e: merged b and d) TMV-positive RNA (red, B-c, T-RNA,) was rarely located on the ER (B-f: merged c and d), but it appeared in the nucleus. (C) Neither CP nor TMV-positive RNA was found in cells transfected with PBS (C-c) (scale bar: 10 μm).

We next used an unrelated probe as a negative control to verify the location of TMV-positive RNA. Cells transfected with TMV-RNA for 24 and 48 h were fixed and analysed using the same IF and RNA-FISH techniques the only difference was that instead of the ER staining step, cell nuclei were stained with DAPI (blue, Invitrogen). Thus blue fluorescence represented the location of the nucleus in Figure 5. When we monitored the cells after TMV-RNA transfection for 24 h, TMV-positive RNA (red) was found throughout the cells as small, irregularly-sized granules (Figure 5B, H.P.T. 24 h) that then accumulated in the nucleus as high-density granules after 48 h (Figure 5C, H.P.T. 48 h). In addition, CP (green) was located around the nucleus and accumulated in the cytoplasm within 48 h. In contrast, TMV-positive RNA could not be detected in cells transfected with TMV-RNA that were hybridized with an unrelated probe (Figure 5A-c, Con). The above results suggest that TMV-RNA uses intracellular ribosomes to synthesize viral proteins in the ER and that the positive RNA localizes to the nucleus to perform other functions. This result might explain our previous result (Figure 3B-b) that showed the presence of suspected TMV virions in HeLa cells.

Accumulation of TMV-positive RNA in HeLa cell nuclei

IF and RNA-FISH were combined to verify the location of TMV-positive RNA. HeLa cells were transfected with TMV-RNA for 24 (A, B, H.P.T. 24 h) or 48 h post-transfection (C, H.P.T. 48 h), and then fixed and hybridized with an unrelated probe (A, Con) or a strand-specific probe (B, C). Except for the blue fluorescence indicating the nucleus (stained with DAPI, A-b, B-d, C-d, blue) instead of the ER (Figure 4), the other stains were the same as in Figure 4(A), (A–C), HeLa cells, DIC (differential interference contrast) CP (B-b, C-b, green) was consistently found in the cytoplasm within 48 h (B-e, C-e, merged b and d). At 24 H.P.T., TMV-positive RNA (B-c, T-RNA, red) was dispersed throughout the cell, and it accumulated largely around the nucleus (B-f, merged c and d). As the time was prolonged to 48 h, TMV-positive RNA (C-c, T-RNA, red) accumulated in HeLa cell nuclei (C-f, merged c and d). This result suggested that TMV-positive RNA was gradually transported from the cytoplasm into the nucleus (especially accumulating in the nucleolus), whereas it was not found in cells transfected with TMV-RNA but instead hybridized with an unrelated probe labelled with the Cy3 fluorochrome (A-c) (scale bar: 10 μm).

IF and RNA-FISH were combined to verify the location of TMV-positive RNA. HeLa cells were transfected with TMV-RNA for 24 (A, B, H.P.T. 24 h) or 48 h post-transfection (C, H.P.T. 48 h), and then fixed and hybridized with an unrelated probe (A, Con) or a strand-specific probe (B, C). Except for the blue fluorescence indicating the nucleus (stained with DAPI, A-b, B-d, C-d, blue) instead of the ER (Figure 4), the other stains were the same as in Figure 4(A), (A–C), HeLa cells, DIC (differential interference contrast) CP (B-b, C-b, green) was consistently found in the cytoplasm within 48 h (B-e, C-e, merged b and d). At 24 H.P.T., TMV-positive RNA (B-c, T-RNA, red) was dispersed throughout the cell, and it accumulated largely around the nucleus (B-f, merged c and d). As the time was prolonged to 48 h, TMV-positive RNA (C-c, T-RNA, red) accumulated in HeLa cell nuclei (C-f, merged c and d). This result suggested that TMV-positive RNA was gradually transported from the cytoplasm into the nucleus (especially accumulating in the nucleolus), whereas it was not found in cells transfected with TMV-RNA but instead hybridized with an unrelated probe labelled with the Cy3 fluorochrome (A-c) (scale bar: 10 μm).

TMV-RNA induced ERS

The initial goal of the ERS response is to protect stressed cells by re-establishing homoeostasis or otherwise neutralizing the damaging consequences of an insult. Herein, we found that TMV-RNA promoted the accumulation of CP in the ER. We therefore hypothesized that the accumulation of this protein induced ERS in HeLa cells.

To test this hypothesis, we assessed the expression of GRP78, a marker for ERS, in both whole-cell and ER fractions. Western blotting results indicate that, within 24 h, GRP78 accumulated both in the whole-cell and ER fractions, and its level markedly increased when the transfection time was extended to 72 h. In contrast, no accumulation of GRP78 was observed in HeLa cells not transfected with TMV-RNA (Figure 6A). These data indicate that TMV-RNA induces ERS the accumulation of TMV protein in the ER induces ERS and activates GRP78 to bind to unfolded proteins (such as CP), thereby increasing their folding capacity. Once the abundance of these unfolded proteins exceeds the capacity of GRP78, ERS would further trigger a series of events, such as autophagy, to restore normal cell function.

GRP78, a marker of ERS, is activated and accumulates in the ER, and electron micrographs show that the formation of autophagosomes is associated with expansion of the ER membrane

(A) Western blotting for GRP78 using lysates from HeLa cells [transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h] or the ER fraction of HeLa cells (transfected with TMV-RNA for 24, 48 or 72 h) indicated that GRP78 was highly expressed in HeLa cells and accumulated mainly in the ER when the transfection time was extended to 72 h. β-Actin was used as a loading control. (H.P.T., hours post-transfection). (B) Representative transmission electron micrographs depicting the ultrastructure of HeLa cells transfected with either PBS (a, Con) or TMV-RNA for 48 h. Analysis of these micrographs revealed that most of the ER membranes in TMV-RNA-transfected cells were in a state of expansion, and numerous vacuoles, including nascent vacuoles and typical autophagosomes with double-membrane vacuolar structures containing visible cytoplasmic contents, appeared around the expanded ER. The distal end of the ER membrane expanded to resemble swollen fingers or forks, which was seldom observed in controls (a). As shown in the images, the distal end of the ER membrane expanded towards the anterior vacuole (b, box) An ER membrane that expanded to resemble swollen fingers might have just sequestered a vacuole (c, box, arrowheads) another distal ER membrane end expanded towards the anterior vacuole and appeared to encapsulate it (d, box). The other distal end of the expanded ER membrane just sequestered a vacuole (d, arrowheads), and the ER membrane was so extremely expanded that it might have produced more vacuoles (e, arrowheads). Nascent vacuoles emerging from the end point of expanded ER membranes (f, box, arrowheads) and fusion events might have occurred between lysosomes and autophagic vacuoles (f, box). Finally, a huge autolysosome was formed in the cytoplasm, partial ER membranes expanded to resemble forks (g, two small boxes), and one vacuole was just sequestered from other partial ER membranes (g, arrowheads) (scale bar: 2 μm). From these micrographs, we presume that ERS stimulated the expansion of the ER, resulting in the formation of autophagosomes.

(A) Western blotting for GRP78 using lysates from HeLa cells [transfected with PBS (Con) or TMV-RNA for 24, 48 or 72 h] or the ER fraction of HeLa cells (transfected with TMV-RNA for 24, 48 or 72 h) indicated that GRP78 was highly expressed in HeLa cells and accumulated mainly in the ER when the transfection time was extended to 72 h. β-Actin was used as a loading control. (H.P.T., hours post-transfection). (B) Representative transmission electron micrographs depicting the ultrastructure of HeLa cells transfected with either PBS (a, Con) or TMV-RNA for 48 h. Analysis of these micrographs revealed that most of the ER membranes in TMV-RNA-transfected cells were in a state of expansion, and numerous vacuoles, including nascent vacuoles and typical autophagosomes with double-membrane vacuolar structures containing visible cytoplasmic contents, appeared around the expanded ER. The distal end of the ER membrane expanded to resemble swollen fingers or forks, which was seldom observed in controls (a). As shown in the images, the distal end of the ER membrane expanded towards the anterior vacuole (b, box) An ER membrane that expanded to resemble swollen fingers might have just sequestered a vacuole (c, box, arrowheads) another distal ER membrane end expanded towards the anterior vacuole and appeared to encapsulate it (d, box). The other distal end of the expanded ER membrane just sequestered a vacuole (d, arrowheads), and the ER membrane was so extremely expanded that it might have produced more vacuoles (e, arrowheads). Nascent vacuoles emerging from the end point of expanded ER membranes (f, box, arrowheads) and fusion events might have occurred between lysosomes and autophagic vacuoles (f, box). Finally, a huge autolysosome was formed in the cytoplasm, partial ER membranes expanded to resemble forks (g, two small boxes), and one vacuole was just sequestered from other partial ER membranes (g, arrowheads) (scale bar: 2 μm). From these micrographs, we presume that ERS stimulated the expansion of the ER, resulting in the formation of autophagosomes.

ERS is the likely trigger for autophagy

Autophagy is beginning to be recognized as an important player in the life-and-death decisions of the ERS response. The control mechanisms of these processes are not fully understood and are the focus of many ongoing investigations. Several reports have shown that ERS can activate autophagy, and conversely, blocking autophagy can enhance ERS-induced cell death [33]. In recent immunoelectron microscopy studies, Dunn has reported that autophagosomes originating from pre-existing ER acquire lysosomal markers in a stepwise fashion during maturation into autolysosomes. Dunn also classified vacuoles morphologically and cytochemically into two groups: (i) nascent autophagic vacuoles (or autophagosomes) and (ii) degradative autophagic vacuoles (or autolysosomes) [34,35]. Herein, we showed that the invasion of TMV-RNA not only triggered autophagy, but also induced ERS in HeLa cells. To further elucidate the connection between autophagy and ERS, we examined the morphological changes of the cells using EM.

Initially, the EM analysis suggested that the autophagosomal membrane might originate from the ER membrane at the ultrastructural level. As shown in Figure 6(B), most of the ER membranes in TMV-RNA-transfected cells were in a state of expansion, and numerous vacuoles, including nascent vacuoles and typical autophagosomes with a double-membrane vacuolar structure containing visible cytoplasmic contents, appeared around the expanded ER. The nascent vacuoles appeared to be produced from the ends of ER membranes (arrowheads), and they expanded to resemble swollen fingers in some pictures. Such structures might be transported to form autophagosomes. Some images also demonstrated that the ER membrane expanded to resemble forks extending towards the anterior vacuole (Figures 6B-b, 6B-d and 6B-g), suggesting that partial ER membranes might be used to form phagophores, structures that will fuse with a nascent vacuole. Neither autophagic vacuoles nor the expansion of the ER was found in cells not transfected with TMV-RNA (Figure 6B-a). Taken together, there are a series of interesting events that are occurring the distal end of the ER membrane expanded like swollen fingers or forks, which might indicate that the cells were stimulated by the TMV-RNA and induced into a state of ERS. This phenomenon of ER membrane expansion was not found in cells not transfected with TMV-RNA (Figure 6B-a).

As shown, the ER membrane resembling swollen fingers might have just produced a vacuole (Figure 6B-c, arrowheads) the distal ER membrane end expanded to resemble forks that extend towards the anterior vacuole (Figure 6B-b, pane) and another distal ER membrane end expanded towards the anterior vacuole and appeared to encapsulate it (Figure 6B-d). Moreover, the other distal end of the expanded ER membrane was sequestered in a vacuole (Figure 6B-d, arrowheads). The ER membrane expanded so greatly that it might have produced more vacuoles (Figure 6B-e, arrowheads), with nascent vacuoles emerging from the end points of the expanded ER membranes (Figure 6B-f, arrowheads). Moreover, fusion events might have occurred between lysosomes and autophagic vacuoles (Figure 6B-f, pane). Finally, a huge autolysosome formed in the cytoplasm, some partial ER membranes expanded to resemble forks extending towards the vacuole (Figure 6B-g, two small panes) and one vacuole had just been produced from other partial ER membranes (Figure 6B-g, arrowheads). Based on these data, it appeared that the process of autophagy, including the maturation and formation of autophagosomes, was required for the expansion of the ER membrane. Hence all of these observations confirmed that the membranes of TMV-RNA-related autophagic vacuoles might be contiguous with or originate from ER membranes. This result explains why more and more vacuoles appeared and accumulated around the expanded ER membranes when the transfection time was extended. We presume that, under ERS, the distal end of the ER membrane expands to maximize the transformation into more vacuoles, which when encapsulated by a monolayer membrane, are called phagophores (which might form from partially expanded ER membranes see Figures 6B-c, 6B-d and B-f). When expanded phagophore cisterns wrap around a portion of the cytoplasm and/or organelles, autophagy is triggered. During the autophagosomal maturation process, the segregated cytoplasm that is engulfed by a phagophore is delivered to the endo/lysosomal lumen by fusion events between autophagosomes and endosomal and/or lysosomal vesicles. Both the cytoplasm and the surrounding membrane are then degraded by lysosomal hydrolases, and the degradation products are transported back to the cytoplasm where they can be re-used for metabolism. Together with our data, the expansion of the ER induced by ERS results in the formation of autophagosomes in TMV-RNA-transfected HeLa cells.

These studies provide some clues into the pathway of TMV-RNA-induced autophagy. Once the TMV-RNA enters HeLa cells, it first synthesizes proteins in the ER, as evidenced by CP in the ER. With the accumulation of TMV proteins in the ER, cells experience ERS and activate ER chaperones (such as GRP78 in the ER lumen) to increase protein folding capacity. If enough TMV proteins are produced so that the capacity of GRP78 is exceeded, ERS stimulates the ends of ER membranes to expand to maximize sequestration into vacuoles in which TMV proteins (such as CP) might be produced. These new vacuoles are wrapped rapidly by phagophores (which also likely originate from the ER and look like the forks in our electron micrographs) that further form autophagosomes. Finally, with the fusion of autophagosomes and lysosomes, autolysosomes form to digest the vacuolar contents we suggest that these vacuolar contents might not be digested, but instead, immune escape occurs through some unknown molecular mechanism. The latter assertion is supported by our evidence of the accumulation of TMV proteins on and around autophagosomal membranes, by the increased CP in the ER and by the enrichment of TMV-positive RNA in the nucleolus. In conclusion, TMV-RNA induces ERS-related autophagy in HeLa cells, and a potential defence mechanism involving ERS and ERS-related autophagy utilized by HeLa cells against TMV-RNA is modelled in Figure 7.

Schematic diagram of the potential ERS-related autophagic defence mechanism utilized by HeLa cells against TMV-RNA

After TMV-RNA enters HeLa cells, it is translated into protein by ribosomes located on the ER membrane. TMV proteins, such as CP, will likely be recognized as foreign or unfolded proteins in the ER lumen and trigger ERS. GRP78 is a marker of ERS, is highly expressed and acts as a chaperone to assist in the folding of CP. If the quantity of CP is too great, GRP78 might leave CP and stimulate the distal end of ER membranes to expand and swell to sequester CP into vacuoles. These new vacuoles would be enwrapped rapidly by phagophores, which likely originate from the ER. These new phagophores resemble forks extending towards the vacuole, and they engulf it to promote maturation into double-membrane autophagosomes. Finally, by fusion with a lysosome, autophagosomes become mono-membrane-structured autolysosomes. The majority of the CP load is degraded. In an alternative pathway, CP might not be degraded and instead achieves immune escape through some unknown molecular mechanism.

After TMV-RNA enters HeLa cells, it is translated into protein by ribosomes located on the ER membrane. TMV proteins, such as CP, will likely be recognized as foreign or unfolded proteins in the ER lumen and trigger ERS. GRP78 is a marker of ERS, is highly expressed and acts as a chaperone to assist in the folding of CP. If the quantity of CP is too great, GRP78 might leave CP and stimulate the distal end of ER membranes to expand and swell to sequester CP into vacuoles. These new vacuoles would be enwrapped rapidly by phagophores, which likely originate from the ER. These new phagophores resemble forks extending towards the vacuole, and they engulf it to promote maturation into double-membrane autophagosomes. Finally, by fusion with a lysosome, autophagosomes become mono-membrane-structured autolysosomes. The majority of the CP load is degraded. In an alternative pathway, CP might not be degraded and instead achieves immune escape through some unknown molecular mechanism.


MATERIALS AND METHODS

Plant Materials, Virus Strains, and Virus Inoculations

Nicotiana tabacum and Nicotiana benthamiana were grown as described ( Harries et al., 2008). Purified virus ( TMV) U1 strain, ( Shintaku et al., 1996)] or virus from plant extracts TVCV, CMV, and PVX ( Yang et al., 2004) were used in some studies. Plant extracts containing TVCV, CMV, or PVX were obtained by grinding infected leaf tissue in liquid nitrogen followed by addition of approximately 2 volumes of 0.1 m sodium phosphate buffer (pH 7). Crude extracts or purified TMV U1 strain diluted with phosphate buffer, then were inoculated directly to plants dusted with carborundum (330 grit, Fisher), as described ( Ding et al., 1998). Additional studies were conducted using plasmids encoding TMV. MP.GFP. CP (previously referred to as TMV- MP-GFP- CP), TMV. MP-GFP (previously referred to as TMV- MP:GFP), and TMV. MP-GFP. CP (previously referred to as TMV- MP:GFP- CP Cheng et al., 2000 Liu et al., 2005), and TVCV-GFP, TBSV-GFP, and PVX-GFP (sources described in Harries et al., 2009). Viral infectious transcripts were obtained from 1 µg linearized plasmid DNA using appropriate T7/T3/SP6 mMessage mMachine in vitro transcription kit (Ambion). Plant leaves were rub-inoculated mechanically with one-half of each transcript reaction using carborundum as an abrasive. Virus-inoculated plants were either placed in the growth chamber or greenhouse. Growth chamber conditions were 23°C +/− 1°C with 16 h of light (approximately 155 µmol m –2 s −1 ) and 8 h of dark. Greenhouse conditions were 24°C +/− 2°C with 16 h supplemental lighting (400 µmol m –2 s −1 ) and 60% humidity.

Isolation and Purification of TMV Replicase Complex

The TMV RNA replicase-containing fractions were purified from infected N. tabacum ‘Xanthi-nn’ as described ( Osman and Buck, 1997). Leaves from N. tabacum plants inoculated with TMV (U1 strain) were harvested at 4 dpi and homogenized in buffer B. The TMV replicase complex was further isolated by differential centrifugation to give a 30,000g pellet, which was further subjected to a Suc density gradient (20% to 60% [wt/wt] in Tris-EDTA-dithiothreitol (TED) buffer) centrifugation. TED buffer consisted of 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 5% (vol/vol) glycerol. Suc density gradient fractions were analyzed for RNA POL activity as described by Osman and Buck (1997) with some modifications discussed below. Active fractions (fractions 4 and 5 from the top) were chosen for further purification. Those were diluted 10-fold with TED buffer, centrifuged at 40,000g, and the resulting pellet containing crude membrane-bound RNA POL was solubilized with sodium taurodeoxycholate and subjected to anion-exchange chromatography on DEAE-Bio-Gel A (Bio-Rad) and High Q (Bio-Rad) using an HPLC system (Pharmacia) as described previously ( Osman and Buck, 1997). Collected fractions were then assayed for RNA POL activity. The reactions were performed using 50 µL of RNA POL preparation added to 50 µL 2× buffer B containing 2 m m ATP, 2 m m GTP, 2 m m CTP, 20 µ m UTP, 10 µCi [α- 32 P]UTP, and bentonite (4.8 mg/mL). Reactions were carried out for 1 h at 30°C. Reaction products were isolated by phenol extraction and ethanol precipitation and resuspended in 10 µL of Tris-EDTA buffer (10 m m Tris-HCl, pH 8.0, 1 m m EDTA) and analyzed by PAGE containing 8M urea followed by autoradiography. Fractions showing highest RNA POL activity were analyzed for protein composition using 4% to 15% SDS-PAGE, followed by protein staining with the Bio-Rad Silver Stain Plus kit. Similar procedures were performed using tissue from uninfected plants to obtain control samples. Excision, in-gel tryptic digests, and mass spectrometry analyses of individual protein bands were conducted as described ( Watson et al., 2003).

Crude VRC fractions were obtained as described ( Covey and Hull, 1981) with the following modifications. Tissue was extracted at 4 dpi with TMV (U1 strain) expressing an MP-GFP fusion and CP when the virus was in maximum replication phase and the MP-GFP was present in the VRC, thus providing a marker for this complex ( Liu et al., 2005). Leaves were ground in liquid nitrogen, followed by addition of buffer A (50 m m Tris-HCl, pH 7.6, 60 m m KCl, 6m m 2-mercaptoethanol, and complete EDTA-free protease inhibitor [Roche]) at 4°C. Extract was then filtered through eight layers of cheese cloth. The filtrate was subjected to centrifugation at 2,000g for 10 min at 4°C. The pellet fraction was resuspended in buffer A containing Triton X-100 (1%) and EDTA (5 m m ) and repelleted by centrifugation at 2,000g for 10 min. The centrifugation process above was repeated two times and the final pellet resuspended in buffer A containing glycerol (15%). The purified TMV VRCs were analyzed for fluorescence from GFP through confocal microscopy. Proteins within fractions enriched for GFP, were separated by SDS-PAGE ( Laemmli, 1970) and visualized through silver staining as described ( Blum et al., 1987). Excision, in-gel tryptic digests, and nano- LC-MS/MS analyses of individual protein bands were conducted as described ( Lei et al., 2005). Protein identification was performed by searching against the National Center for Biotechnology Information nonredundant protein database.

VIGS Constructs and Agroinfiltration

Full-length AtpC and RCA complementary DNA ( cDNA) sequences from N. tabacum were amplified by RT-PCR using Moloney murine leukemia virus reverse transcriptase (Promega), exTaq POL (Takara), oligo dT, and gene specific primers ( Supplemental Table S1 ). The full-length cDNAs were cloned into pCR-Blunt II-TOPO as instructed (Invitrogen). A 425-bp fragment from the open reading frame of AtpC and RCA were amplified by PCR using exTaq POL and gene specific primers ( Supplemental Table S1 ), sequenced, and cloned into the silencing vector, pTRV2 ( Liu et al., 2002), using Gateway cloning technology (Invitrogen). pTRV1 and pTRV2 constructs in Agrobacterium tumefaciens were grown and infiltrated into N. benthamiana leaves as described ( Ding et al., 2004). Gene silencing was confirmed by standard RNA extraction and quantitative RT-PCR analysis.

Total RNA Extraction and Quantitative RT-PCR Analysis

Total RNA from leaf tissues were extracted using an RNeasy plant mini kit (Qiagen) and treated with DNase I. First-strand cDNA was synthesized using a 12- to 18-base oligo(dT) primer (Invitrogen) and Moloney murine leukemia virus reverse transcriptase (Promega). Two microliters of 20-fold diluted cDNA, gene specific primers, and the Power SYBR Green master mix (Applied Biosystems) were used for quantitative RT-PCR analyses of AtpC and Rca mRNA levels with an ABI Prism 7900 HT sequence detection system (Applied Biosystems). To normalize the mRNA levels of target genes between samples, relative EF1α mRNA levels were determined using EF1α-specific primers and a relative quantification method ( Pfaffl, 2001). The influence of virus infection on EF1α and ubiquitin mRNA levels also was determined through this method. Additionally, virus levels in tissues were determined through reverse transcription and quantitative PCR with virus-specific primers. To create standard curves used to quantify virus levels, virus RNA was isolated from infected tissue through virus-specific procedures: TMV and TVCV ( Bruening et al., 1976 Gooding and Hebert, 1967), PVX ( AbouHaidar et al., 1998 Francki and McLean, 1968), and CMV ( Roossinck and White, 1998). The viral RNA was quantified by spectrophotometry. Viral RNA levels from treatment tissue were quantified by comparing their cycle threshold values obtained during quantitative RT-PCR with cycle threshold values obtained from mock-inoculated tissue spiked with known amounts of viral RNA. Reverse-strand synthesis utilized SSRT III (Invitrogen). Primers for all quantitative RT-PCR reactions are provided ( Supplemental Table S1 ).

Statistical analysis of VRC numbers and sizes in AtpC- and Rca-silenced compared with mock- and TRV-infected control plants utilized generalized linear or linear mixed models (number: GENMOD procedure, size: MIXED procedure) with repeated measurements in SAS 9.3 (SAS Institute, Inc.). As necessary, the data were transformed to meet assumptions for equal variances between means. Individual treatment mean comparisons utilized the Tukey-Kramer adjustment of P values.

ELISAs

For ELISA analyses, discs of equal size containing tissue from outside the perimeter of the largest green fluorescent lesion were harvested at a particular dpi, as described in the results, and frozen in liquid nitrogen. Frozen tissues were ground in 100 μL of phosphate-buffered saline ( PBS) buffer (0.14 m NaCl, 3 m m KCl, 10 m m phosphate buffer, pH 7.0), and ELISAs were performed for TMV CP accumulation as described ( Derrick et al., 1997).

GST Constructs and Pull-Down Assay

The cDNA fragments of MT (nucleotides 69–959), I, II (nucleotides 956–2487), HEL (nucleotides 2483–3416), and POL (nucleotides 3421–4916) domains of 126-/183-kD proteins were cloned into pGEX-5X-2 (GE Lifesciences) and expressed in Escherichia coli. Expressed GST or GST-fused MT, I-II, HEL, and POL domains were bound to 50 μl of glutathione sepharose 4B as instructed (GE Lifesciences). Supernatant fraction (S30) from N. tabacum was prepared as described previously ( Yamaji et al., 2006). Tubes containing approximately equal amounts of GST or GST-virus gene fusions bound to sepharose beads, as determined through SDS-PAGE analysis of the bound beads, were incubated with 200 μL of S30 fraction in protein interaction buffer ( PBS containing 1% Triton X-100, 0.5% NP-40, 0.1% SDS, and 1m m phenylmethylsulfonyl fluoride) for 1 h at 4°C. The beads were washed three times, twice with PBS containing 1% Triton X-100 and once in PBS. Unbound fractions were removed and the bound beads were heated in SDS-PAGE sample buffer at 100°C for 10 min and centrifuged at 18,000g for 5 min. The supernatant was resolved on 4% to 10% SDS-PAGE and subjected to immunoblotting.

Total Protein Extraction and Western-Blot Assays

Plant tissue frozen in liquid nitrogen was ground to fine powder and thawed in protein extraction buffer (5 m m Tris-HCl, pH 8.0, 150 m m NaCl, 2 m m MgCl2, 50 m m KCl, 1% Triton X-100, 0.5 m m dithiothreitol) containing protease inhibitor (cOmplete EDTA-free, Roche 1 tablet/50 mL). Samples were placed on ice for 30 min and centrifuged at 18,000g for 10 min. Fifteen micrograms of each protein sample was boiled in SDS-PAGE sample buffer at 100°C for 10 min and centrifuged at 18,000g for 5 min. The supernatant was resolved on 4% to 10% SDS-PAGE and electroblotted to polyvinylidene difluoride membranes (Immun-Blot, Bio-Rad). Blots were blocked with Tris-buffered saline containing 5% skim milk and probed with the appropriate primary antibodies from rabbits, anti-RCA (1:7,000 dilution), or anti-GFP (0.5 μg/mL BioVision Research Products). They were then washed in Tris-buffered saline buffer containing 0.05% Tween 20 and probed with anti-rabbit alkaline phosphatase-conjugated secondary antibody (Promega). The immunoprobed proteins were detected by colorimetric reaction using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (Promega).

Microscopy

Fluorescent viral lesion images were captured with an Olympus SZX-12 fluorescent stereomicroscope (Olympus). Lesion areas (pixels) were measured using the MetaMorph 4.5 program (Universal Imaging). TMV. MP-GFP VRCs were imaged on a Bio-Rad model 1024ES. Images were processed using Adobe Photoshop. GFP was excited using a 488-nm line from a krypton/argon laser and emissions were captured at 522 nm.

Subcellular Localization of AtpC and RCA

Full length AtpC and Rca open reading frames were cloned into 5G-mCherry C1 plasmid, obtained from Dr. Elison Blancaflor (Noble Foundation) and introduced into A. tumefaciens GV2260 by electroporation. For studies on the interaction of AtpC and RCA with ectopically expressed 126-kD protein-GFP fusion, A. tumefaciens containing the 126-kD protein-GFP fusion was coinfiltrated with A. tumefaciens containing AtpC-mCherry or Rca-mCherry using a needleless syringe and observed 2 dpi. For studies involving later challenge with the virus, N. benthamiana leaves were syringe-infiltrated with A. tumefaciens containing AtpC-mCherry or Rca-mCherry and placed in the growth chamber for 2 d. Leaves expressing AtpC-mCherry or RCA-mCherry then were inoculated with TMV. MP-GFP, and 4 dpi, the infected cells were imaged using a Perkin-Elmer UltraView ERS spinning-disc confocal system coupled to a Zeiss Observer D1 inverted microscope. The cells were observed with a 63× water-immersion objective. GFP was excited at 488 nm, and emission was detected at 510 nm. Chloroplast autofluorescence was detected by excitation at 647 nm, and emission was detected at 680 nm. mCherry was excited at 587 nm, and emission was detected at 610 nm.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers X63606 and Z14980.

Supplemental Data

Supplemental Figure S1 . Fraction analysis for TMV 126- and 183-kD proteins and RNA POL activity.

Supplemental Figure S2 . Lack of AtpC- or RCA-mCherry colocalization with TMV.MP-GFP in epidermal cells of N. benthamiana.

Supplemental Figure S3 . Lack of AtpC- or RCA-mCherry colocalization with a 126-kD protein-GFP fusion in epidermal cells of N. benthamiana.

Supplemental Figure S4 . Host EF1α and ubiquitin mRNA levels in N. tabacum challenged with TVCV, CMV, and PVX.

Supplemental Figure S5 . Virus accumulation in extracts from challenged plants analyzed for AtpC and Rca mRNA levels in Figure 2.

Supplemental Figure S6 . AtpC and Rca mRNA silencing phenotypes in N. benthamiana plants.

Supplemental Figure S7 . Influence of AtpC or Rca silencing on intercellular movement of TBSV-GFP and PVX-GFP.

Supplemental Table S1 . Primers for VIGS constructs and quantitative RT-PCR analyses.


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