Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication.

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

A Viral Suppressor Modulates the Plant Immune Response Early in Infection by Regulating MicroRNA Activity.

Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant's immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR's miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro Most importantly, in contrast to controls with a Δp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.IMPORTANCE Plant viruses manipulate their hosts in various ways. Viral suppressor proteins (VSRs) interfere with the plant's immune response by sequestering small, antivirally acting vsiRNAs, which are processed from viral RNAs during the plant's RNA-silencing response. Here, we examined the effects of VSRs on cellular microRNAs (miRNAs), which show a high degree of similarity with vsiRNAs. Binding experiments with two unrelated VSRs and three important regulatory miRNAs revealed that the proteins exhibit similar miRNA-binding profiles but bind different miRNAs at considerably different affinities. Most interestingly, experiments in plants showed that in the early infection phase, the Tombusvirus VSR p19 modulates the activity of these miRNAs on their target mRNAs very differently and that this differential regulation strictly correlates with the binding affinities of p19 for the respective miRNAs. Our data suggest that VSRs may specifically control plant gene expression and the early immune response by differential sequestration of miRNAs.

Co-opting ATP-generating glycolytic enzyme PGK1 phosphoglycerate kinase facilitates the assembly of viral replicase complexes.

The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment.

The involvement of ROS producing aldehyde oxidase in plant response to Tombusvirus infection.

The influence of Tomato bushy stunt virus (TBSV) infection on the activity and isoformic composition of aldehyde oxidase and catalase in Nicotiana benthamiana plants was investigated. It was shown that the infection of plants with TBSV results in enhancement of leaf aldehyde oxidase (AO) isoforms AO2 and AO3. Significantly enhanced levels of superoxide radical producing activity of AO isoforms were also detected. This is the first demonstration of involvement of plant AO in defense mechanisms against viral infection. In addition, the infection caused an increased accumulation of hydrogen peroxide, compared to mock-inoculated plants. The virus infection resulted in increased activity of catalase (CAT) and superoxide dismutase (SOD) in roots and leaves of N. benthamiana. Moreover, activation of two additional CAT isoforms was observed in the leaves of plants after virus inoculation. Our findings indicate that the virus infection significantly affects enzymes responsible for the balance of ROS accumulation in plant tissue in response to pathogen attack.

RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection.

UNLABELLED: The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, the MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if the MOI is controlled by virus functions, we estimated the MOI in plants coinfected by two genetic variants of Tomato bushy stunt virus (TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusvirus, Cymbidium ringspot virus (CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (~4.0) than in TBSV-TBSV or TBSV-DI-RNA coinfections (~1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different virus-encoded functions. IMPORTANCE: The number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, the MOI may have an optimum value that maximizes virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants coinfected by two genotypes of Tomato bushy stunt virus (TBSV), the MOI was lower than in plants coinfected by TBSV and Cymbidium ringspot virus (CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.

A high-throughput approach for studying virus replication in yeast.

Viruses are intracellular pathogens that are dependent on viral and host factors for multiplication. Model hosts, such as yeast, can be very valuable in identifying host factors involved in viral replication. Yeast is also useful for studies on functional interactions of host factors with viral proteins and/or virus nucleic acids. The advantages of using yeast include the availability of a single gene-deletion library and the essential gene library (yTHC); the controllable small- or large-scale expression of viral proteins and nucleic acids; and the rapid growth of yeast strains. Procedures that facilitate high-throughput analysis of host factors and plant and animal RNA virus replication in yeast, with a plant virus (tombusvirus; TBSV) and an animal virus (nodavirus; FHV) as examples, are described.

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing.

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 degrees C, but not at 33 degrees C. By contrast, systemic infection at 33 degrees C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 degrees C, but not at 33 degrees C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.

Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes.

Plus-strand RNA virus replication takes place on distinct membranous surfaces in infected cells via the assembly of viral replicase complexes involving multiple viral and host proteins. One group of tombusviruses, such as Tomato bushy stunt virus (TBSV), replicate on the surfaces of peroxisomal membranes in plant and yeast cells. Surprisingly, previous genome-wide screen performed in yeast demonstrated that a TBSV replicon RNA replicated as efficiently in yeast defective in peroxisome biogenesis as in the wt yeast (Panavas et al., Proc Natl Acad Sci U S A, 2005). To further test how the lack of peroxisomes could affect tombusvirus replication, we used yeast cells missing either PEX3 or PEX19 genes, which are absolutely essential for peroxisome biogenesis. Confocal microscopy-based approach revealed that the wild-type tombusvirus p33 replication protein accumulated in the endoplasmic reticulum (ER) in pex3Delta or pex19Delta yeast, suggesting that tombusvirus replication could take place on the surface of ER membrane. The activities of the isolated tombusvirus replicase preparations from wt, pex3Delta or pex19Delta yeasts were comparable, demonstrating that the assembly of the replicase was as efficient in the ER as in the authentic subcellular environments. The generation/accumulation of tombusvirus recombinants was similar in wt, pex3Delta and pex19Delta yeasts, suggesting that the rate of mistakes occurring during tombusvirus replication is comparable in the presence or absence of peroxisomes. Overall, this work demonstrates that a tombusvirus, relying on the wt replication proteins, can efficiently replicate on an alternative intracellular membrane. This suggests that RNA viruses might have remarkable flexibility for using various host membranes for their replication.

A novel plant homeodomain protein interacts in a functionally relevant manner with a virus movement protein.

Tomato bushy stunt virus and its cell-to-cell movement protein (MP; P22) provide valuable tools to study trafficking of macromolecules through plants. This study shows that wild-type P22 and selected movement-defective P22 amino acid substitution mutants were equivalent for biochemical features commonly associated with MPs (i.e. RNA binding, phosphorylation, and membrane partitioning). This generated the hypothesis that their movement defect was caused by improper interaction between the P22 mutants and one or more host factors. To test this, P22 was used as bait in a yeast (Saccharomyces cerevisiae) two-hybrid screen with a tobacco (Nicotiana tabacum) cDNA library, which identified a new plant homeodomain leucine-zipper protein that reproducibly interacted with P22 but not with various control proteins. These results were confirmed with an independent in vitro binding test. An mRNA for the host protein was detected in plants, and its accumulation was enhanced upon Tomato bushy stunt virus infection of two plant species. The significance of this interaction was further demonstrated by the failure of the homeodomain protein to interact efficiently with two of the well-defined movement-deficient P22 mutants in yeast and in vitro. This is the first report, to our knowledge, that a new plant homeodomain leucine-zipper protein interacts specifically and in a functionally relevant manner with a plant virus MP.

Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus.

Post-transcriptional gene silencing (PTGS) is a sequence-specific degradation mechanism that operates in almost all eukaryotic cells. In plants, double-stranded RNA triggers PTGS, generating 21- to 25-nucleotide guide RNAs responsible for specific degradation of cognate mRNA. The double stranded RNA intermediates of replicating plant viruses often induce PTGS, leading to symptom attenuation. Here we demonstrate the role of PTGS in defective interfering (DI) RNA-mediated symptom attenuation in plants infected with Cymbidium ringspot tombusvirus (CymRSV). Analysis of 21- to 25-nucleotide RNAs in Nicotiana benthamiana infected with CymRSV indicated that PTGS was not spread homogeneously along the viral genome. The 21- to 25-nucleotide RNAs derived mainly from plus-stranded RNA and likely arose from local basepaired structures. In contrast to helper viral RNA, short DI RNAs were not accessible to helper virus-induced RNA degradation guided by the 21- to 25-nucleotide RNAs. Our results suggest a model in which PTGS plays an important role in the selective accumulation and symptom attenuation mediated by DI RNAs. Because PTGS operates in a wide variety of different organisms, this model is applicable to DI RNA generation and accumulation in both plant and animal cells.

Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene.

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

De novo generation and accumulation of tomato bushy stunt virus defective interfering RNAs without serial host passage.

Studies were initiated to monitor generation and accumulation of defective interfering (DI) RNAs associated with tomato bushy stunt virus (TBSV) in the absence of serial, high multiplicity of infection passage. Infections were initiated in Nicotiana clevelandii host plants and protoplast cell suspensions by inoculation with in vitro-synthesized infectious TBSV RNA transcripts containing a genomic marker. The infections were then assayed for DI-size RNAs by both Northern blot analysis and reverse transcription coupled with PCR amplification. DI-size RNAs could not be detected by Northern blot analysis in either plants or protoplasts after an evident viral infection. However, RT-PCR amplification permitted the isolation of DI-size cDNAs (600-700 nt) from plant, but not protoplast, infections as early as 8 days postinoculation. Sequence analysis of these DI-size cDNA clones revealed that they contained the four conserved regions found in all previously identified competent DI RNAs. Several DI RNA clones contained the genomic marker which confirmed their de novo generation from the input transcript inoculum. A comparison of the nucleotide sequence of these clones to previously sequenced DI RNAs, isolated from plants after multiple passages, showed that differences existed at the junctions between regions. These results demonstrate that a heterogeneous population of DI RNAs accumulated in plants in the absence of serial host passage. In addition, the similarity of these DI RNAs to previously characterized DI RNAs that accumulate upon passage indicates that evolution can occur very rapidly within the initially inoculated plant.