The Rabies Virion‐Associated 100‐kDa Polypeptide (VAP100) Is a Host‐Derived Minor Component of the Viral Envelope

Abstract

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virusinfected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.

Further Characterization of a CD99‐Related 21‐kDa Transmembrane Protein (VAP21) Expressed in Syrian Hamster Cells and Its Possible Involvement in Vesicular Stomatitis Virus Production

“…Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed using the Laemmli's discontinuous buffer system (10). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 (CBB) or subjected to the immunoblot analysis as previously described (25). In brief, cells were rinsed twice with PBS(Ϫ) and lysed in a sample lysis buffer [62.5 mM TrisHCl, 2% SDS, 4% 2-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue; (pH 6.8)].…”

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We have reported that, in addition to actin (13), several other host-derived components are also incorporated into the rabies virions as minor but regular constituents, including actin-related ERM family proteins (ezrin, radixin and moesin; 18), actin-anchoring transmembrane protein (24), a constitutive type of heat shock protein (Hsc73; 15), and fibronectin and integrin (our unpublished data). In addition, we recently found that other cellular components, VAP21 (21-kDa) and VAP100 (100-kDa), also are incorporated into the rabies virion grown in a Syrian baby hamster kidney cell line, 17,25). We also have shown that the VAP21 is a CD99-related 21-kDa transmembrane protein and is incorporated into the virion of many kinds of other enveloped viruses grown in BHK-21 cells (26).

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“…In addition, we recently found that other cellular components, VAP21 (21-kDa) and VAP100 (100-kDa), also are incorporated into the rabies virion grown in a Syrian baby hamster kidney cell line, 17,25). We also have shown that the VAP21 is a CD99-related 21-kDa transmembrane protein and is incorporated into the virion of many kinds of other enveloped viruses grown in BHK-21 cells (26).…”