Coupling of rotavirus genome replication and capsid assembly.
ABSTRACT The Reoviridae family represents a diverse collection of viruses with segmented double-stranded (ds)RNA genomes, including some that are significant causes of disease in humans, livestock, and plants. The genome segments of these viruses are never detected free in the infected cell but are transcribed and replicated within viral cores by RNA-dependent RNA polymerase (RdRP). Insight into the replication mechanism has been provided from studies on Rotavirus, a member of the Reoviridae whose RdRP can specifically recognize viral plus (+) strand RNAs and catalyze their replication to dsRNAs in vitro. These analyses have revealed that although the rotavirus RdRP can interact with recognition signals in (+) strand RNAs in the absence of other proteins, the conversion of this complex to one that can support initiation of dsRNA synthesis requires the presence and partial assembly of the core capsid protein. By this mechanism, the viral polymerase can carry out dsRNA synthesis only when capsid protein is available to package its newly made product. By preventing the accumulation of naked dsRNA within the cell, the virus avoids triggering dsRNA-dependent interferon signaling pathways that can induce expression and activation of antiviral host proteins.
- Future Virology 03/2009; 4(2):185-196. · 1.00 Impact Factor
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ABSTRACT: Study of the classical swine fever virus (CSFV) replication is challenging because it is a BSL-3-Ag pathogen that requires specialized facilities. We developed a cell-based assay in human embryonic kidney 293T cells that can quantify the activities of NS5B, the CSFV RNA-dependent RNA polymerase. The 5BR assay uses transiently-expressed CSFV NS5B to produce RNAs that activate the RIG-I-mediated signaling pathway to result in reporter protein production. Upon co-expression of the CSFV core protein, we observed enhancement of the CSFV RdRp activity. The CSFV core and NS5B proteins could co-immunoprecipitate with each other and co-localize in cells, when visualized by confocal microscopy. Analyses of combinations of RdRps and capsid proteins from different viruses demonstrated that the CSFV core could enhance the CSFV NS5B activity in a virus species-specific manner. Studies of truncated versions of CSFV core demonstrated that the first 30 residues of core protein are dispensable for interaction with the CSFV NS5B. Purified core protein could enhance RNA synthesis by the purified NS5B in vitro, with the increase being in the synthesis of the de novo-initiated RNA. These results demonstrate that the CSFV core protein can regulate the mechanism of RNA synthesis by the CSFV RdRp.Virus Genes 05/2014; 49(1). · 1.84 Impact Factor
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ABSTRACT: The rotavirus (RV) genome consists of eleven segments of double-stranded (ds)RNA. Typically, each segment contains 5' and 3' untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a single protein. RV variants have been described with segments of atypical size owing to sequence rearrangements. In many cases, the rearrangement originates from a partial head-to-tail sequence duplication that initiates after the stop codon of the ORF, leaving the protein product of the segment unaffected. To probe the limits of the RV genome to accommodate additional genetic sequence, we used reverse genetics to insert duplications (analogous to synthetic rearrangements) and heterologous sequences into the 3' UTR of the segment encoding NSP2 (gene 8). The approach allowed the recovery of recombinant RVs that contained sequence duplications (up to 200 bp) and heterologous sequences including those for FLAG, the hepatitis C virus E2 epitope, and the internal ribosome entry site of cricket paralysis virus. The recombinant RVs grew to high titer (>10(7) PFU/ml) and remained genetically stable during serial passage. Despite their longer 3' UTRs, rearranged RNAs of recombinant RVs expressed wildtype levels of protein in vivo. Competitive growth experiments indicated that, unlike RV segments with naturally occurring sequence duplications, genetically engineered segments were packaged less efficiently into progeny viruses. Thus, features of naturally occurring rearranged segments, other than their increased length, contribute to their enhanced packaging phenotype. Our results define strategies for developing recombinant RVs as expression vectors, potentially leading to next-generation RV vaccines that induce protection against other infectious agents.Journal of Virology 03/2013; · 4.65 Impact Factor