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, Val37lle 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.

Pathogenesis of Semliki Forest Virus Encephalitis

“…Several series of SFV mutants, including temperature-sensitive (ts) mutants, have been described (Tan et al, 1969;Keranen and Kaariainen, 1974;Hearne et al, 1987;Barrett et al, 1980). A number of strains including prototype, which is related to L10, A7, and A7(74), have been cloned and sequenced (Garoff et al, 1980;Takkinen, 1986;Glasgow et al, 1994;Santagati et al, 1995Santagati et al, , 1998Tarbatt et al, 1997;Tuittila et al, 2000). The molecular clone of the prototype strain, known as SFV4, has been engineered to form a much used viral expression and vaccine system (Liljestrom and Garoff, 1991;Tubulekas et al, 1997).…”

The Pathogenesis of Alphaviruses

“…e A7 strain has been sequenced and the sequence compared to the more virulent prototype strain [63][64][65][66]. One of the most marked features of the A7 sequence is the presence of a long untranslated sequence containing multiple repeats at the 3 � end of the genome.…”

“…is region is 334 nucleotides longer than in the prototype strain. However, it has no in�uence on virulence [64]. ere are multiple mutations throughout the A7 genome compared to the prototype strain, and a large proportion result in amino acid substitutions.…”

Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence

“…Infection with the SFV A7(74) strain is lethal only for mice younger than 2 weeks, whereas in older mice, virus spread is severely restricted in CNS neurons and infection remains asymptomatic (Fazakerley et al, 1993;Oliver et al, 1997); transient demyelination of axons is observed occasionally (Amor et al, 1996;Pathak et al, 1983). In a number of alphaviruses, mutations introduced into the glycoproteins alter virulence; for example, a derivative of virulent SFV4 expressing the A7(74) E2 glycoprotein is apathogenic for adult mice (Santagati et al, 1995) and single amino acid mutations in the E2 protein attenuate the virus (Santagati et al, 1998). However, as shown by our previous experiments, replacement of the entire structural region of SFV4 with that of A7(74) yields a fully lethal virus, while a reciprocal SFV recombinant is avirulent , thus proving the importance of the nonstructural protein genes for virulence.…”