Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals

Olmsted, Robert A.; Meyer, William J.; Johnston, Robert E.

doi:10.1016/0042-6822(86)90322-3

Cited by 64 publications

(57 citation statements)

References 13 publications

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“…Furthermore, the finding of a widely distributed HS-binding site on E2 is compatible with the fact that the E2c epitope is conformational and covers a large stretch of E2. Binding of MAb R6 to this epitope is affected by mutations in residues 62, 96, 114, and 159, which are distant from each other in the primary protein sequence but are likely to be located near each other in the three-dimensional folded structure of E2 (37,39).…”

Section: Discussionmentioning

confidence: 99%

Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Byrnes¹,

Griffin

2000

J Virol

114

101

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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.The alphaviruses are RNA viruses which are carried by hematophagous insects, such as mosquitoes, and can infect a wide variety of mammalian and avian hosts. In vertebrates, they can replicate extremely rapidly and cause high-titer viremia, which allows transmission of the virus to new mosquitoes. Sindbis virus (SV) is a particularly well-studied alphavirus which causes a mild rash and arthritis in humans but can cause fatal encephalomyelitis in mice.The cell surface receptors which allow alphaviruses such as SV to infect such a broad variety of species have not yet been conclusively determined, but it has recently been shown that SV can attach to heparan sulfate (HS), a negatively charged glycosaminoglycan expressed on many types of cells (4,23,32). Sulfated glycosaminoglycans on the cell surface and in the extracellular matrix normally bind a wide variety of growth factors, chemokines, enzymes, and matrix components (30, 45) but are also important in the attachment of a number of bacteria, protozoa, and viruses (42). Proteins typically bind electrostatically to HS by use of stretches of positively charged amino acids such as Lys and Arg, and attachment of SV to HS is presumably mediated in the same fashion. Although the use of cell surface HS greatly increases the efficiency of SV attachment, it is not absolutely required for infection, and a distantly related Alphavirus, Ross River virus, does not bind HS at all (4). It has been proposed that the abil...

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Section: Discussionmentioning

confidence: 99%

Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Byrnes¹,

Griffin

2000

J Virol

114

101

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“…E2-a is defined by MAbs 49 and 50 (Stec et al, 1986). These MAbs compete not only with MAbs to this site but also with MAbs (R8/10) defining site E2-b (Olmsted et al, 1986), which is independently mutable, suggesting that E2-a and E2-b overlap. MAbs 49 and R10 both compete strongly (>90%) with our MAb 202, suggesting that the latter also identifies the E2-ab site.…”

Section: Discussionmentioning

confidence: 99%

“…The SV E2 MAbs reported originally by Schmaljohn et al (1983) did not identify distinct epitopes, although functional analysis of anti-E2 MAbs by other investigators suggested that distinct epitopes were likely to be present (Roehrig et al, 1980;Stanley et al, 1985Stanley et al, , 1986. Further studies of reactivity of MAbs with different strains of SV (Olmsted et al, 1986;David et al, 1987) and analysis of neutralization escape mutants (Stec et al, 1986) has led to identification of three different regions of antibody reactivity on E2. E2-a is defined by MAbs 49 and 50 (Stec et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

Monoclonal Antibodies to the E1 and E2 Glycoproteins of Sindbis Virus: Definition of Epitopes and Efficiency of Protection from Fatal Encephalitis

Mendoza

Stanley

Griffin

1988

Journal of General Virology

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SUMMARYProtection of mice from fatal neuroadapted Sindbis virus encephalitis can be accomplished by passive transfer of monoclonal antibodies (MAbs) to either the E1 or E2 glycoprotein of Sindbis virus. Both neutralizing and non-neutralizing MAbs can be protective. To define further the characteristics of MAbs that provide protection from fatal disease, antigenic epitopes on the E1 and E2 glycoproteins were identified using a competitive binding enzyme immunoassay. Four distinct epitopes on E1 and three on E2 were defined. MAbs to all E1 epitopes, both neutralizing (three) and nonneutralizing (one) protected mice from fatal encephalitis. MAbs to the E2 neutralizing epitopes (two) protected mice from fatal encephalitis while those to the nonneutralizing epitope did not. The efficiency of protection from fatal Sindbis virus encephalitis of four neutralizing and non-neutralizing protective anti-E1 and anti-E2 MAbs representing different epitopes was compared. The neutralizing MAbs (against epitopes E2-ab, E2-c and El-c) gave 50% protection at lower doses (2 to 20 gg) than the non-neutralizing MAb representing epitope El-e (150 gg) when given before virus challenge. When given after virus challenge, MAbs to E2-ab and E2-c protected at lower doses (0,03 to 0.3 Ixg) than did either MAbs to El-c (> 100 gg) or El-e (10 p~g). The MAbs to El-e, E2-ab and E2-c were required in larger amounts to afford protection before than after challenge, while the opposite was true for MAb to E 1-c.

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“…The rate of virus penetration into cells was measured in terms of the rate at which resistance to neutralization by added polyclonal antibody was acquired by the infecting virus (Olmsted et al, 1986 ;Vrati et al, 1996). Briefly, PS monolayers in 60 mm dishes were infected with approximately 100 p.f.u.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth.

Vrati

Agarwal

Malik

et al. 1999

Journal of General Virology

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The biological properties of an Indian isolate (GP78) of Japanese encephalitis virus (JEV) were characterized in tissue-cultured cells and mice and these were compared with the JaOArS982 strain from Japan. The GP78 strain had a markedly extended lag phase during its growth in porcine stable kidney (PS) cells. There were no obvious defects in the penetration of GP78 into PS cells. However, viral RNA and protein synthesis were significantly delayed in GP78-infected PS cells. Fusion-from-within assays carried out in C6/36 cells indicated that GP78 was less fusogenic than the JaOArS982 strain of JEV. Moreover, maximum fusion in GP78-infected cells occurred at pH 5n5, whereas JaOArS982-infected cells showed maximum fusion at pH 6n0. These results suggested that there may be a lesion in the virus-cell fusion process. The GP78 strain also showed delayed growth in brains of 1-week-old BALB/c mice. Although JEV GP78 was as virulent as the JaOArS982 strain in these mice, the appearance of clinical symptoms of JEV infection was delayed by a day in mice infected with the GP78 strain and these animals showed an increased average survival time.Comparison of the nucleotide sequences of the GP78 and the JaOArS982 strains of JEV identified a number of amino acid substitutions in structural proteins. Of these, a Thr Met substitution at residue 76 of the envelope protein is predicted to be causally associated with the altered biology of the GP78 strain during growth.

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Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals

Cited by 64 publications

References 13 publications

Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Monoclonal Antibodies to the E1 and E2 Glycoproteins of Sindbis Virus: Definition of Epitopes and Efficiency of Protection from Fatal Encephalitis

Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth.

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