Dataset Information

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

ABSTRACT: The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.

SUBMITTER: Lattwein E

PROVIDER: S-EPMC3255886 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

Lattwein E E Klemens O O Schwindt S S Becher P P Tautz N N

Journal of virology 20111026 1

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to proce ...[more]

PMID: 22031952

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Project description:Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins.IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.

Project description:In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase) in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase) of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase) and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C(ATPase) coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C(ATPase) is an essential component of the replication complex, and (iv) 2C(ATPase) has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.

Dataset Information

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

Publications

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

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