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E G Strauss

Publications and source records attributed to E G Strauss.

At least 19 recordsLinked to original sources
PubMed1992

Identification of the active site residues in the nsP2 proteinase of Sindbis virus.

The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.

Amino Acid Sequence↗
PubMed1992

High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells.

Sindbis virus is an alphavirus with a very wide host range, being able to infect many birds and mammals as well as mosquitoes. We have isolated a monoclonal antibody that largely blocks virus binding to mammalian cells. This antibody was found to be directed against the C-terminal domain of the high-affinity laminin receptor, a 67-kDa protein present on the cell surface that binds with high affinity to basement membrane laminin and that is known to be important in development and in tumor invasion. This receptor is believed to be formed from a 295-amino-acid polypeptide that is modified in some unknown way after translation. The primary sequence of this 295-amino-acid protein is highly conserved among mammals. We found the hamster amino acid sequence to be identical to a mouse sequence and to differ at only two amino acids from a human sequence and at two amino acids from a bovine sequence. To verify the importance of the laminin receptor for infection by Sindbis virus, hamster cells were stably transfected with the gene encoding the 295-amino-acid protein under the control of a high-efficiency promoter. Such transfected hamster cells overexpressed the laminin receptor at the cell surface, bound severalfold more Sindbis virions than did the parental cells, and became infected by Sindbis virus with a higher efficiency. In contrast, cells transfected with the antisense gene expressed less laminin receptor on the surface and were less susceptible to the virus. Binding of the virus varied linearly with the amount of laminin receptor on the cell surface, whereas infectivity measured with a plaque assay varied with the 1.4 power of the receptor concentration, suggesting that interaction with more than one receptor aids virus penetration. By these criteria, the laminin receptor functions as the major receptor for Sindbis virus entry into mammalian cells. We also found that the anti-laminin receptor antibody partially blocked Sindbis virus binding to mosquito cells, suggesting that the laminin receptor is conserved in mosquitoes and functions as a Sindbis virus receptor in this host. The wide distribution of this highly conserved receptor may be in part responsible for the broad host range exhibited by the virus, which infects a wide range of mammals and birds as well as its mosquito vector and can infect many different tissues within these hosts.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Sindbis virus RNA polymerase is degraded by the N-end rule pathway.

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met greater than Ala greater than Tyr greater than or equal to Phe greater than Agr. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires read-through of an opal termination codon such that it is underproduced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.

Amino Acid Sequence↗
PubMed1991

Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses.

Ockelbo virus was first isolated in 1982 in Sweden. It is the causal agent of disease in humans characterized by arthritis, rash, and fever and is antigenically very closely related to Sindbis virus. We have determined the nucleotide and translated amino acid sequences of the prototype Ockelbo virus isolate (82-5) to determine the relatedness of Ockelbo virus to Sindbis virus at the genomic level and clarify the taxonomic position of Ockelbo virus within the alphavirus genus. The numbers of nucleotides and of translated amino acids in each region of the Ockelbo virus genome were exactly the same as those for the prototype AR339 strain of Sindbis virus except for three small deletions and insertions in the C-terminal half of nsP3 and for three single nucleotide insertions and deletions in the 3' untranslated region. Overall there were 672 nucleotide differences (5.7% divergence), resulting in 97 amino acid changes (2.6% divergence), between the two viruses: more than 85% of the nucleotide changes were silent. Only the C-terminal domain of nsP3 and the E2 glycoprotein showed a higher degree of amino acid substitution than the overall average. The former domain is not conserved among alphaviruses, and the latter is primarily responsible for antigenic variation. Sequence analysis of 420 nucleotides at the 3' end of a number of other Sindbis-like alphaviruses, including Karelian fever virus and South African, Indian, and Australian isolates of Sindbis virus, demonstrated that Ockelbo virus is more closely related to South African strains of Sindbis virus than it is to the prototypic Egyptian AR339 strain. Thus the South African strains, which have caused epidemic disease in humans, may have been introduced into Northern Europe by man or by migratory birds to establish Ockelbo disease there. The Indian and Australian strains form a distinct branch of the evolutionary tree and differ from prototypic AR339 Sindbis virus in 17% of the nucleotides sequenced.

Amino Acid Sequence↗
PubMed1991

RNA viruses: genome structure and evolution.

The explosive pace of sequencing of RNA viruses is leading to rapid advances in our understanding of the evolution of these viruses and of the ways in which their genomes are organized and expressed. New insights are coming not only from genomic nucleotide sequence comparisons, but also from direct sequencing of transcribed mRNAs and of RNAs that serve as intermediates in replication.

Biological Evolution↗
PubMed1991

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.

Amino Acid Sequence↗
PubMed1990

Complete sequence of the genomic RNA of O'nyong-nyong virus and its use in the construction of alphavirus phylogenetic trees.

The alphaviruses are a group of about 25 positive-strand RNA viruses that are important human and veterinary pathogens and that are geographically dispersed. We report here the complete nucleotide sequence of the genomic RNA of the alphavirus, O'nyong-nyong virus. The RNA is 11,835 nucleotides in length and the organization of the genome is typical of alphaviruses. Phylogenetic trees were constructed from the protein sequences of O'nyong-nyong and six other alphaviruses. Trees were constructed for each nonstructural and structural viral protein individually in order to detect any possible recombination events, as well as to examine the differential divergence among the various proteins. The members of each tree can be divided into three subgroups: the Semliki Forest virus subgroup (Semliki Forest, O'nyong-nyong, and Ross River viruses), the eastern equine encephalitis virus subgroup (eastern equine encephalitis and Venezuelan equine encephalitis viruses), and the Sindbis virus subgroup. Sindbis virus, which is geographically restricted to the Old World, is more closely related to the eastern equine encephalitis subgroup, which are New World viruses, than it is to the Semliki Forest virus subgroup, which are mostly Old World viruses. Western equine encephalitis virus is a special case because it is a recombinant virus. Its nonstructural and capsid proteins are most closely related to those of eastern equine encephalitis virus while its glycoproteins are most closely related to those of Sindbis virus. All members of a given subgroup have diverged the same amount from their common node point. However, the structural proteins of the Semliki Forest virus subgroup are more closely related to one another than those of the eastern equine encephalitis virus subgroup. This difference probably indicates that the members of the eastern equine encephalitis virus subgroup diverged earlier than the members of the Semliki Forest virus subgroup, which suggests that the alphaviruses originated in the New World.

Alphavirus↗
PubMed1990

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus.

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.

Animals↗
PubMed1989

Low pH-dependent Sindbis virus-induced fusion of BHK cells: differences between strains correlate with amino acid changes in the E1 glycoprotein.

Expression of alphavirus glycoproteins on the surface of infected cells leads to cell fusion after exposure to acidic pH. Two strains of Sindbis virus, AR339 (SV) and neuroadapted Sindbis virus (NSV), which differ in virulence for weanling mice, were found to differ in pH-dependent fusion. BHK-21 cells infected with SV fused maximally after shifting to pH 5.4, whereas cells infected with NSV required a lower pH, pH 4.8, for maximal fusion. No difference was noted in the optimal pH for agglutination of goose erythrocytes (5.75 for both viruses). To determine the molecular basis for the difference in fusion a series of recombinant viruses was constructed using a cDNA clone of Sindbis virus from which infectious RNA can be transcribed in vitro. Cells infected with a recombinant virus that had the SV E1 and NSV E2 genes had a fusion response curve as a function of pH like SV, while cells infected with recombinant virus with the NSV E1 and SV E2 genes fused like NSV. The E1 glycoproteins of SV and NSV differ at two positions: Val-72 in SV is Ala in NSV (a change near the putative fusion site), and Gly-313 in SV is Asp in NSV. Recombinant viruses which had Val-72 (SV) and Asp-313 (NSV) or Ala-72 (NSV) and Gly-313 (SV) had a lowered pH of fusion like NSV suggesting that both positions participate in determining some aspect of the conformational change in the E1-E2 heterodimer associated with pH-dependent fusion.

Animals↗
PubMed1989

Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4.

Temperature-sensitive (ts) mutants of Sindbis virus belonging to complementation group F, ts6, ts110, and ts118, are defective in RNA synthesis at the nonpermissive temperature. cDNA clones of these group F mutants, as well as of ts+ revertants, have been constructed. To assign the ts phenotype to a specific region in the viral genome, restriction fragments from the mutant cDNA clones were used to replace the corresponding regions of the full-length clone Toto1101 of Sindbis virus. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious transcripts, and the virus recovered was tested for temperature sensitivity. After the ts lesion of each mutant was mapped to a specific region of 400 to 800 nucleotides by this approach, this region of the cDNA clones of both the ts mutant and ts+ revertants was sequenced in order to determine the precise nucleotide change and amino acid substitution responsible for each mutation. Rescued mutants, which have a uniform background except for one or two defined changes, were examined for viral RNA synthesis and complementation to show that the phenotypes observed were the result of the mutations mapped. ts6 and ts110 had a single base substitution in nsP4, resulting in replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this, together with the fact that all RNA synthesis in ts6-infected cells and, to a lesser extent, in ts110-infected cells shut off when the cells were shifted from a permissive to a nonpermissive temperature, suggests that nsP4 is the virus polymerase. ts118 was a double mutant. It contained a single base substitution in nsP2, resulting in replacement of Val by Ala at position 425 that resulted in the formation of minute plaques, but not in a reduction in the plaque number at the nonpermissive condition. The second change, a substitution of Gln by Arg in ts118 at residue 93 in nsP4, had little apparent phenotype on its own, but in combination with the change in nsP2 led to a ts phenotype. Thus, in each case the mutation responsible for the temperature sensitivity of the three known complementation group F mutants lay in nsP4. In addition, the result with ts118 suggests that nsP2 and nsP4 may interact with each other in a complex.

Amino Acid Sequence↗
PubMed1989

Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins.

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved.

Amino Acid Sequence↗
PubMed1989

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions.

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.

Animals↗
PubMed1988

Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses.

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.

Amino Acid Sequence↗
PubMed1988

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Alphavirus↗
PubMed1988

Western equine encephalitis virus is a recombinant virus.

The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.

Amino Acid Sequence↗
PubMed1988

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.

Age Factors↗