Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell.

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.     

Heterologous movement protein strongly modifies the infection phenotype of cucumber mosaic virus

A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.     

Ilarviruses Encode a Cucumovirus-Like 2b Gene That Is Absent in Other Genera within the Bromoviridae

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.     

Satellite RNA reduces expression of the 2b suppressor protein resulting in the attenuation of symptoms caused by Cucumber mosaic virus infection

Satellite RNAs (satRNAs) depend on cognate helper viruses for replication, encapsidation, movement and transmission. Many satRNAs with different symptom modulation effects have been reported. The pathogenicity of satRNAs is thought to be the result of a direct interaction among the satRNA, helper viruses and host factors by unknown mechanisms. To understand the effect of satRNA of Cucumber mosaic virus (a severe field ShanDong strain, SD-CMV) on pathogenicity, and the possible involvement of host RNA silencing pathways in pathogenicity, we constructed biologically active CMV cDNA clones and a CMV-Δ2b mutant lacking the open reading frame of 2b, a silencing suppressor protein, in order to infect Nicotiana benthamiana and Arabidopsis with or without SD-satRNA. We found that SD-satRNA reduced the accumulation of the 2b protein and its coding RNA4A and attenuated the yellowing caused by SD-CMV infection. Small RNA analysis indicated that the 2b protein interfered with RNA silencing, specifically in the synthesis of CMV RNA3-derived small interfering RNAs (R3-siRNAs). The accumulation of R3-siRNAs in CMV-Δ2b infection was reduced in the presence of satRNA, for which greater accumulation of satRNA-derived siRNAs (satsiRNAs) was detected. Our results suggest that abundant SD-satRNA serving as target for RNA silencing may play a role in protecting helper CMV RNA, especially, subgenomic RNA4, from being targeted by RNA silencing. This compensates for the increase in RNA silencing resulting from the reduction in expression of the 2b suppressor in the presence of satRNA. Our data provide evidence that a plant silencing mechanism is involved in the pathogenicity of satRNA.     

Template-Independent Repair of the 3′ End of Cucumber Mosaic Virus Satellite RNA Controlled by RNAs 1 and 2 of Helper Virus

RNA viruses which do not have a poly(A) tail or a tRNA-like structure for the protection of their vulnerable 3′ termini may have developed a different strategy to maintain their genome integrity. We provide evidence that deletions of up to 7 nucleotides from the 3′ terminus of cucumber mosaic cucumovirus (CMV) satellite RNA (satRNA) were repaired in planta in the presence of the helper virus (HV) CMV. Sequence comparison of 3′-end-repaired satRNA progenies, and of satRNA and HV RNA, suggested that the repair was not dependent on a viral template. The 3′ end of CMV satRNA lacking the last three cytosines was not repaired in planta in the presence of tomato aspermy cucumovirus (TAV), although TAV is an efficient helper for the replication of CMV satRNA. With use of pseudorecombinants constructed by the interchange of RNAs 1 and 2 of TAV and CMV, evidence was provided that the 3′-end repair was controlled by RNAs 1 and 2 of CMV, which encode subunits of the viral RNA replicase. These results, and the observation of short repeated sequences close to the 3′ terminus of repaired molecules, suggest that the HV replicase maintains the integrity of the satRNA genome, playing a role analogous to that of cellular telomerases.     

cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.     

Two amino acids near the N‐terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity

Cucumber mosaic virus (CMV) 2b suppresses RNA silencing primarily through the binding of double‐stranded RNA (dsRNA) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N‐terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild‐type plants, but are efficiently rescued in mutant plants defective in RNA‐dependent RNA polymerase 6 (RDR6) and Dicer‐like 4 (DCL4). Moreover, the transgenic plants of 2b, but not 2blm (L15A/M18A), rescue the high infectivity of CMV‐Δ2b through the suppression of antiviral silencing. L15A, M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long dsRNAs, 2blm is significantly compromised in 21‐bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs. In cross‐linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro. Together, these findings suggest that L15 and M18 of CMV 2b are required for high affinity to ds‐siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.     

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.     

Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b’s efficient suppression of RNA silencing

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.     

Satellite RNA-mediated reduction of cucumber mosaic virus genomic RNAs accumulation in Nicotiana tabacum

Satellite RNAs(satRNAs)are molecular parasites that interfere with the pathogenesis of thehelper viruses.In this study,the relative accumulation of cucumber mosaic virus(CMV)-Fny genomicRNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR.The results showed thatsatRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumu-lation of CMV-Fny genomic RNAs,tested as open reading frames.The accumulation of CMV-Fny 1a,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at differentinoculation times.CMV-FnyΔ2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the2a gene were deleted,caused only a slight mosaic effect on N.tabacum seedlings,similar to that of CMV-Fsat,but the addition of satRs to CMV-FnyΔ2b showed further decrease in the accumulation of CMV-FnyΔ2b genomic RNAs.Our results indicated that the attenuation of CMV,by adding satRs or deleting the2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction ofCMV genomic RNAs accumulation in N.tabacum was possibly related to the 2b gene.     

Cucumber mosaic virus 2b proteins inhibit virus-induced aphid resistance in tobacco

Cucumber mosaic virus (CMV), which is vectored by aphids, has a tripartite RNA genome encoding five proteins. In tobacco (Nicotiana tabacum), a subgroup IA CMV strain, Fny-CMV, increases plant susceptibility to aphid infestation but a viral mutant unable to express the 2b protein (Fny-CMV∆2b) induces aphid resistance. We hypothesized that in tobacco, one or more of the four other Fny-CMV gene products (the 1a or 2a replication proteins, the movement protein, or the coat protein) are potential aphid resistance elicitors, whilst the 2b protein counteracts induction of aphid resistance. Mutation of the Fny-CMV 2b protein indicated that inhibition of virus-induced resistance to aphids (Myzus persicae) depends on amino acid sequences known to control nucleus-to-cytoplasm shuttling. LS-CMV (subgroup II) also increased susceptibility to aphid infestation but the LS-CMV∆2b mutant did not induce aphid resistance. Using reassortant viruses comprising different combinations of LS and Fny genomic RNAs, we showed that Fny-CMV RNA 1 but not LS-CMV RNA 1 conditions aphid resistance in tobacco, suggesting that the Fny-CMV 1a protein triggers resistance. However, the 2b proteins of both strains suppress aphid resistance, suggesting that the ability of 2b proteins to inhibit aphid resistance is conserved among divergent CMV strains.     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

The 2b protein of cucumoviruses has a role in promoting the cell-to-cell movement of pseudorecombinant viruses

Pseudorecombinant viruses (i.e., those containing a reassorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.