RESUMO
Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing â¼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (â¼0.5 kb) derived from the 3' untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5'-3' exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3' UTR. Mutations or deletions of various secondary structures in the 3' UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV(KUN)) 3' UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.
Assuntos
RNA Viral/biossíntese , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/patogenicidade , Regiões 3' não Traduzidas , Animais , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Flavivirus/genética , Flavivirus/patogenicidade , Camundongos , Conformação de Ácido Nucleico , RNA Viral/químicaRESUMO
The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.
Assuntos
Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Metiltransferases , RNA Polimerase Dependente de RNA , Proteínas não Estruturais Virais/imunologia , Vírus do Nilo Ocidental , Animais , Sítios de Ligação , Ligação Competitiva , Feminino , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/imunologia , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Conformação Proteica , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/imunologia , RNA Polimerase Dependente de RNA/metabolismo , Recombinação Genética , Proteínas não Estruturais Virais/síntese química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/enzimologia , Vírus do Nilo Ocidental/imunologiaRESUMO
Transgenic Nicotiana benthamiana plants expressing an untranslatable version of the coat protein (CP) gene from the Tamarillo mosaic virus (TaMV) were either resistant to TaMV infection or recovered from infection. These phenotypes were the result of a post-transcriptional gene silencing (PTGS) mechanism that targeted TaMV-CP sequences for degradation. The TaMV-CP sequences were degraded when present in the wild-type TaMV potyvirus, in transgene mRNA, or in chimeric viral vectors based on White clover mosaic virus. The more efficiently targeted region was mapped to a 134-nt segment. Differences were observed in the efficiency of targeting during cell-to-cell and long-distance movement of the chimeric viruses. However, the TaMV-CP sequences do not appear to be targeted for degradation when delivered by biolistics.
