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.Semliki Forest virus (SFV) is an enveloped positive-stranded RNA virus of the family Togaviridae. The SFV prototype was isolated in 1942 from a pool of mosquitoes in Uganda (40). The A7(74) strain (4) is a derivative of an SFV strain isolated from mosquitoes in Mozambique in 1959 (26). Several strains of SFV, such as L10 (4) and SFV4 (24), cause lethal encephalitis in mice of all ages, leading to death of the animals in a few days (3, 11), whereas A7(74) infection of adult mice is asymptomatic per se but leads to axonal demyelination mediated by CD8 ϩ T cells (2). However, the severity of A7(74) infection is strictly age dependent, being lethal for neonatal mice less than 2 weeks old (8, 27), probably because the strain is capable of virion formation in propagating neurons, unlike in mature neurons (27).SFV nonstructural proteins (nsP) are translated as a polyprotein (nsP1234) from the genomic 42S RNA, and they form essential components of viral RNA replication and transcription complexes (14). The nsP1 protein is a methyl-and guanylyltransferase (1, 18), whereas the nsP2 is a proteinase (45) and nucleoside triphosphatase (33). The nsp3 gene product is a phosphoprotein, and it has been proposed to function together with nsP1 in anchoring the replication complex proteins to cytoplasmic membrane structures (30, 31). In Sindbis virus (SIN), p123 a...

Polyamine-regulated unproductive splicing and translation of spermidine/spermine N1-acetyltransferase

“…Functional titer (PFU, 1.35 3 10 10 ) was determined with plaque assay with an overnight infection in 293 cells. An attenuated strain of Semliki Forest virus SFV A7 (74) was produced and titrated in BHK cells as previously described (Tuittila et al 2000). The titer of the virus stock was 1.2 3 10 9 plaque forming units /mL.…”

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models

“…In this study, the avirulent SFV strain A7(74) 16 and the derived EGFP expressing vector VA7-EGFP 8 were used. The VA7-EGFP vector was utilized in cell culture experiments to verify the spreading and oncolysis in human and rat cancer cells, while both the vector and the parental A7(74) virus were used in antitumor animal studies.…”

“…8,16 For virus production, 4 3 10 6 baby hamster kidney (BHK)-21 cells were seeded onto Ø 10-cm cell culture plates. After 24 hr, the cells were transduced with multiplicity of infection (MOI) 1 in total volume of 7 ml.…”

Oncolytic Capacity of Attenuated Replicative Semliki Forest Virus in Human Melanoma Xenografts in Severe Combined Immunodeficient Mice

“…Prompted by the key phenotypic features, we wanted to investigate the oncolytic potential of the avirulent SFV strain A7(74) in cultured cells and in experimental animals. First, in contrast to wild-type SFV, which is highly neurovirulent and lethal for mice of all ages, SFV A7(74) is relatively harmless to normal adult mice and forms only small perivascular foci of infection in the central nervous system (CNS) of these mice (10)(11)(12). However, in the brains of mice younger than 14 days, where neuronal processes are still maturing and active myelination is ongoing, SFV A7(74) is able to replicate and cause cytopathic death of neural cells similar to wild-type SFV (10,13).…”