A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

Abstract

The replicase protein nsP2 of Semliki Forest virus (SFV) has a 648 RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.The molecular and cell biology of the alphavirus Semliki Forest virus (SFV) have been extensively studied, as has the pathogenesis of SFV infection in laboratory mice (6, 10). Generally, the virus is neuroinvasive, and extraneural inoculation results in a high-titer plasma viremia with infection of central nervous system (CNS) cells by virus passage across the bloodbrain barrier (24,25,11,36). Strains of SFV vary in their neurovirulence in adult mice. Irrespective of the route of inoculation, adult mice infected with the L10, V13, or prototype strains or virus derived from the prototype molecular clone SFV4 rapidly succumb to a fulminant encephalitis, whereas adult mice infected with the A7 or A7(74) strain develop a subclinical encephalitis (8,27). The nominally avirulent strains spread more slowly in the CNS, and host responses prevent widespread fatal infection and eventually eliminate infectious virus (7, 11), though they generate lesions of immune-mediated demyelination (12,37). In SJL mice these lesions are active for many months (9, 35). In contrast to their differences in adult mice, all strains of SFV are virulent in embryonic, neonatal, and suckling mice (5,11,13,15). For the adultavirulent strains A7 and A7 (74) there is a sharp age-related transition (22,23). In suckling mouse brain and in culture, many infected brain cells rapidly undergo apoptosis (3, 17), whereas in immunocompromised adult mice, virus can persist in CNS cells without any apparent cell death for many months (4, 12). Age-related neurovirulence may be related to changes in the propensity of CNS cells to undergo apoptosis on infection (2, 3).The SFV4, A7, and A7(74) strains have been cloned and sequenced (14,16,31,32,39,40,41). Between the strains there are multiple changes scattered throughout the genome (41). The difference in virulence between SFV4 and A7 or A7(74) appears to be polygenic, and to date, changes in the E2 gene, the 5Ј untranslated region, and the nsP3 gene have been shown to be determinants of virulence between these particular strains (32,33,40,41). As with all viruses, there will al...

Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection

“…Our experiments provided evidence that nsP2 is one of the proteins that determine the level of IFN-␣/␤ release. These data correlate with recently published results on the replication of Semliki Forest virus mutants in mouse brains (14). These experiments indicated that mutations in a nuclear localization signal of nsP2 either make the virus more sensitive to interferon or lead to more-efficient induction of IFN-␣/␤.…”

Apoptosis induced by Semliki Forest virus is RNA replication dependent and mediated via Bak

“…It has been controversial whether SFV or SIN kills host cells via receptor binding/endocytosis or RNA replication. [25][26][27] By infecting 3T9 mouse embryonic fibroblasts (MEFs) with ultraviolet (UV)-inactivated SFV and monitoring virus uptake, RNA replication and apoptosis induction, we clearly show that SFV requires an intact RNA genome to induce classical features of apoptosis (Supplementary Material and Supplementary Figure 1). Effective apoptosis is mediated via the activation of caspases.…”

“…Point mutations in nsP2 attenuate or suppress both the cytotoxicity and the shut-off of host protein synthesis, and therefore such mutations may be the basis for the development of new, less cytotoxic and non-cytotoxic SFV and SIN vectors. 27,37 However, as nsP2 is also essential for RNA replication, it cannot be discriminated if its cytotoxic action is due to the lack of RNA production, the inhibition of protein synthesis or the processing/activation of initiator or effector caspases. Here we obtained evidence that inhibition of protein synthesis is not sufficient to trigger the apoptotic program.…”

Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes

“…For example, a single amino acid change in (i) the envelope glycoprotein of Sindbis virus or lymphocytic choriomeningitis virus, (ii) the HA protein of avian influenza A virus, or (iii) the nsP2 replicase of Semliki Forest virus (or two amino acid changes in the envelope glycoprotein of dengue virus) changes the severity of neurovirulence outcomes (16,21,30,31,54). The specific role that each BDV gene plays in neurovirulence and BD phenotype remains poorly characterized.…”