The Significance of the 3′-Nontranslated Region and E2 Amino Acid Mutations in the Virulence of Semliki Forest Virus in Mice

Abstract

We have recently shown that the 3′-nontranslated region (3′-NTR) of the avirulent Semliki Forest virus A7(74) [SFVA7(74)] contains a unique sequence of 101 nucleotides and five repetitive nucleotide units whereas the 3′-NTR of the neurovirulent SFV4 has only two repeats. A chimeric virus was constructed by replacing the entire 3′-NTR of the SFV4 clone with the A7(74) 3′-NTR. The hybrid replicated efficiently in the central nervous system (CNS) of adult Balb/c mice and, similarly to SFV4, led to high mortality after intraperitoneal inoculation. In contrast, another chimeric virus, CME2, containing the E2 gene of the avirulent SFVA7(74) virus in the SFV4 clone was recently shown to be avirulent for mice. Several derivatives with single-site or a constellation of amino acid mutations were constructed. Two single-site E2 mutants, Val37Ile and Asn212Ser, displayed an attenuated phenotype in mice with mortality reduced from 90 to 48 and 43%, respectively. None of the multiple site mutants were significantly attenuated. Adult female mice showed a greater resistance to SFV infection than male mice. The SFV hybrid viruses, CM3NTR and CME2, reached the CNS similarly to the parental viruses, but the single-site E2 mutants were only sporadically found in the CNS. We conclude that in mice the 3′-NTR does not play a significant role in the pathogenesis of Semliki Forest virus and that specific E2 amino acid mutations reduce the virulence, especially in female mice. The results additionally suggest that individual amino acid mutations in the E2 glycoprotein affect the efficiency of migration into the CNS.