Flexible filamentous plant viruses are responsible for more than half the viral crop damage in the world, but are also potentially useful for biotechnology. Structural studies began more than 75 years ago but have failed due to the virion’s extreme flexibility. We have used cryo–EM to generate an atomic model for bamboo mosaic virus revealing flexible N– and C–terminal extensions that allow deformation while still maintaining structural integrity.

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3 untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ϳ35 and ϳ55 kDa were specifically cross-linked with RNA elements, whereupon the ϳ35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3 UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3 UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.The replication of plus-strand RNA viruses is arbitrated by virus-specific replicase complexes (RC) (3), which are membrane-associated replication complexes derived from cell organelles. These membrane structures recruit several host proteins, providing a suitable environment for the synthesis of viral RNA. Unraveling the interactions between viruses and their host cells as a function of time could make considerable contributions to our understanding of the dynamics of viral infections. In vitro RNA-dependent RNA polymerase (RdRp) systems are commonly used to analyze the components of host and viral proteins associated with RdRp complexes, as well as for identifying the putative cis-acting elements of the RNA templates (3, 24). Such systems have been established for several plus-strand RNA plant viruses (4,5,10,18,34,35,36,40,44,47), as well as satellite RNAs (satRNAs) (16,19,22,33,41,52,53).Bamboo mosaic virus (BaMV) is a member of the Potexvirus genus containing a single-stranded, positive-sense RNA genome with flexuous rod-shaped morphology. The BaMV genome consists of a 6,366-nucleotide (nt)-long RNA molecule [excluding the 3Ј poly(A) tail] with a 5Ј cap and a 3Ј poly (A) tail. This single-stranded RNA genome encodes five conserved open reading frames (ORFs) coding for polypeptides of 155 kDa (ORF1), 28 kDa (ORF2), 13 kDa (ORF3), 6 kDa (ORF4), and 25 kDa (ORF5), flanked by 5Ј 94-nt and 3Ј 142-nt untr...

Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.To spread throughout hosts, plant viruses have evolved a number of pathways to allow their progeny to pass across plasmodesmata into neighboring cells and travel along the vascular system (8, 26). The virus-encoded movement proteins play a pivotal role through diverse mechanisms in these cellto-cell and vascular transports. Ancillary proteins, for example, the viral coat proteins (CPs) in some cases, and host factors may also participate in these processes. Numerous studies have been conducted to elucidate the movement mechanisms. Many of the results have been summarized in a number of recent reviews (23,28,30). They provided in-depth discussions on issues such as the identification and characterization of the involved viral and host proteins and the transport models for some exemplified viruses, such as Tobacco mosaic virus (TMV) and Potato virus X (PVX). Despite these efforts, many details of the processes remain elusive.Members of the genus Potexvirus have a positive-strand RNA genome that contains five open reading frames (ORFs), a 5Ј methyl cap, and a 3Ј poly(A) tail. ORF1 encodes the viral replication protein, consisting of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain (RdRp) from the N terminus to the C terminus (16,17). The HLD has RNA 5Ј-triphosphatase and nucleoside triphosphatase (NTPase) activities (18). With the co...

BackgroundSatellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.Methodology/Principal FindingsLeaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.Conclusions/SignificanceThe overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.