Structural Protein Relationships Among Eastern Equine Encephalitis Viruses

Abstract

We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the El, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidlymigrating E2-associated protein and a high 3//,. (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates, In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA-and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA-and SAserotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NAand SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.

A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates

“…Western blotting was performed with the sera from mice injected with a WEEV, EEEV, VEEV or purified EEV VLPs and horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). E1 and E2 glycoproteins of WEEV and VEEV VLPs did not separate, probably because of similar molecular weights of the two glycoproteins (difference by 2 to 3 kDa) and heterogeneous posttranslational modifications, such as glycosylation, consistent with the previously published reports (45,46).…”

Structural Plasticity of the Semliki Forest Virus Glycome upon Interspecies Transmission

“…For example, across the eastern equine encephalitis alphaviruses, E1 exhibits complex-type glycosylation, while E2 has a high level of Endo H-sensitive structures. 32 Similarly, Barmah Forest virus exhibits predominantly complex-type glycosylation on E1 but has no glycans on E2. 29 , 30 In Sindbis virus, E1 primarily exhibited complex-type glycosylation, while E2 exhibited a greater degree of oligomannose-type glycosylation.…”

“…Alphaviral glycosylation is an important determinant of cellular tropism and virulence, − and the composition of alphavirion glycans derived from mammalian cell lines has been the subject of extensive study. − The alphaviral E1 and E2 glycoproteins are post-translationally modified with N-linked glycosylation. For example, SFV has one N-linked sequon on E1 and two on E2 .…”

Detection of Antibodies to Alphaviruses and Discrimination between Antibodies to Eastern and Western Equine Encephalitis Viruses in Rabbit Sera using a Recombinant Antigen and Virus‐specific Monoclonal Antibodies

“…Three different serocomplexes represented by eastern equine encephalitis viruses (EEEV), western equine encephalitis viruses (WEEV) and Venezuelan equine encephalitis viruses (VEEV) are found throughout most parts of the New World (Weaver et al, 1999). The EEEV serocomplex can be further classified into North American (NA) and South American (SA) subtypes, which differ in epidemiological, biological and genetic characteristics (Strizki and Repik, 1994;Weaver et al, 1994). Infections with EEEV-SA are often fatal for horses, while human infections are rare and clinically less severe than infections with EEEV-NA which in turn may cause severe diseases in both horses and men, with high mortality rates (Weaver et al, 1999).…”