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Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

ABSTRACT: Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

SUBMITTER: Tuittila MT

PROVIDER: S-EPMC111978 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

Tuittila M T MT Santagati M G MG Röyttä M M Määttä J A JA Hinkkanen A E AE

Journal of virology 20000501 10

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotid ...[more]

PMID: 10775594

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Project description:The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV.SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes were confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.

Project description:RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

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Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

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Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

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