Molecular basis of Sindbis virus neurovirulence in mice.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.     

Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus.

Strains of Sindbis virus differ in their virulence for mice of different ages; this variation is related in large part to variations in the amino acid compositions of E1 and E2, the surface glycoproteins. The comparative pathogenesis of Sindbis virus strains which are virulent or avirulent for newborn mice has not been previously examined. We have studied the diseases caused by a virulent wild-type strain, AR339, and two less virulent laboratory strains, Toto1101 and HRSP (HR small plaque). After peripheral inoculation of 1,000 PFU, AR339 causes 100% mortality within 5 days (50% lethal dose [LD50] = 3 PFU) while Toto1101 causes 70% mortality (LD50 = 10(2.4) PFU) and HRSP causes 50 to 60% mortality (LD50 = 10(5.1) PFU) with most deaths occurring 7 to 11 days after infection. However, after intracerebral inoculation of 1,000 PFU, Toto1101 is virulent (100% mortality within 5 days; LD50 = 4 PFU) while HRSP is not (75% mortality; LD50 = 10(4.2) PFU). After intracerebral inoculation, all three strains initiate new virus formation within 4 h, but HRSP reaches a plateau of 10(6) PFU/g of brain while Toto1101 and AR339 replicate to a level of 10(8) to 10(9) PFU/g of brain within 24 h. Interferon induction parallels virus growth. Mice infected with HRSP develop persistent central nervous system infection (10(6) PFU/g of brain) until the initiation of a virus-specific immune response 7 to 8 days after infection when virus clearance begins. The distribution of virus in the brains of mice was similar, but the virus was more abundant in the case of AR339. HRSP continued to spread until day 9. Clearance from the brain was complete by day 17. We conclude that the decreased virulence of HRSP is due to an intrinsic decreased ability of this strain of Sindbis virus to grow in neural cells of the mouse. We also conclude that CD-1 mice do not respond to the antigens of Sindbis virus until approximately 1 week of age. This lack of response does not lead to tolerance and persistent infection but rather to late virus clearance whenever the immune response is initiated.     

Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.     

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.     

Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome

Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981-1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HR(sp) (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5' nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W.B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 +/- 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 +/- 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 +/- 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.     

Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.     

Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro.

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.     

Adaptation of Sindbis Virus to BHK Cells Selects for Use of Heparan Sulfate as an Attachment Receptor

Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981–1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.     

Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes.

The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.     

The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice.

The E2 glycoprotein of Sindbis virus is synthesized as a precursor, PE2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. The four-residue PE2 cleavage signal conforms to the basic amino acid-X-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). In this report, we present evidence that the amino acid which immediately follows the signal, the N-terminal residue of E2, can influence protease recognition, binding, and/or cleavage of PE2. Constructs encoding nine different amino acids at E2 position 1 (E2 1) were produced by site-directed mutagenesis of the full-length cDNA clone of our laboratory strain of Sindbis virus AR339 (pTRSB). Viruses derived from clones encoding Arg (TRSB), Asp, Ser, Phe, His, and Asn in a nonglycosylated form at E2 1 contained predominantly E2. Viruses encoding Ile, Leu, or Val at E2 1 contained the uncleaved form of PE2. The specific infectivity of TRSB (E2 Arg-1) for baby hamster kidney (BHK-21) cells was from 5- to greater than 100-fold higher than those of isogenic constructs with other residues at E2 1, suggesting that E2 Arg-1 represents a BHK-21 cell adaptive mutation in our laboratory strain. In newborn CD-1 mice, TRSB was more virulent than the PE2-containing viruses but less virulent than other PE2-cleaving viruses with alternative amino acids at E2 1. These results indicate that in TRSB, E2 Arg-1 increased the efficiency of virus-cell interactions in cultured BHK-21 cells but simultaneously decreased the ability of virus to mediate in vivo virus-cell interactions critical for the induction of disease. This suggests that the N terminus of E2 may participate in or be associated with virion domains which mediate these viral functions.     

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.     

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence

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.     

Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.     

Viral determinants of age-dependent virulence of Sindbis virus for mice.

Many alphaviruses cause more severe disease in young animals than in older animals. The age-dependent resistance to severe disease is determined primarily by maturation of the host, but strains of virus can be selected that overcome the increased resistance of mature animals. Sindbis virus (SV) strain AR339 causes fatal encephalitis in newborn mice and nonfatal encephalitis in weanling mice, whereas NSV, a neuroadapted strain of SV, causes fatal encephalitis in weanling as well as newborn mice. We have previously shown that the E2 glycoprotein of NSV contained His-55, whereas AR339 E2 had Gln-55 (S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss, J. Virol. 62:2329-2336, 1988) and that SV with E2 containing Gly-172 was more virulent for newborn mice than SV with E2 containing Arg-172 (P. C. Tucker and D. E. Griffin, J. Virol. 65:1551-1557, 1991). Here we tested the virulence for both newborn and older mice of SV containing a number of different amino acids at E2 position 55 (His, Gln, Lys, Arg, Glu, Gly) in combination with both Gly-172 and Arg-172. All the viruses were virulent for newborn mice, but the residues at both 55 and 172 influenced the virulence of the virus, and there were differences in virulence observed among the various viruses. However, only viruses with His-55 were fully virulent for 14-day-old mice, and this virulence was independent of the residue at position 172. Virus with Lys-55 was virulent for 7-day-old mice, although slightly attenuated relative to His-55. Viruses with His-55 grew more rapidly and to higher titer in the brains of 7- and 14-day-old mice, in N18 neuroblastoma cells, and in BHK cells. Our data suggest that His-55 is important for neurovirulence in older mice and acts by increasing the efficiency of virus replication.     

Identification of genes involved in the host response to neurovirulent alphavirus infection

Single-amino-acid mutations in Sindbis virus proteins can convert clinically silent encephalitis into uniformly lethal disease. However, little is known about the host gene response during avirulent and virulent central nervous system (CNS) infections. To identify candidate host genes that modulate alphavirus neurovirulence, we utilized GeneChip Expression analysis to compare CNS gene expression in mice infected with two strains of Sindbis virus that differ by one amino acid in the E2 envelope glycoprotein. Infection with Sindbis virus, dsTE12H (E2-55 HIS), resulted in 100% mortality in 10-day-old mice, whereas no disease was observed in mice infected with dsTE12Q (E2-55 GLN). dsTE12H, compared with dsTE12Q, replicated to higher titers in mouse brain and induced more CNS apoptosis. Infection with the neurovirulent dsTE12H strain was associated with both a greater number of host genes with increased expression and greater changes in levels of host gene expression than was infection with the nonvirulent dsTE12Q strain. In particular, dsTE12H infection resulted in greater increases in the levels of mRNAs encoding chemokines, proteins involved in antigen presentation and protein degradation, complement proteins, interferon-regulated proteins, and mitochondrial proteins. At least some of these increases may be beneficial for the host, as evidenced by the demonstration that enforced expression of the antiapoptotic mitochondrial protein peripheral benzodiazepine receptor (PBR) protects neonatal mice against lethal Sindbis virus infection. Thus, our findings identify specific host genes that may play a role in the host protective or pathologic response to neurovirulent Sindbis virus infection.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.