Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

Abstract

Murine gammaherpesvirus 68 (MHV-68) glycoprotein B (gB) was identified in purified virions by immunoblotting, immunoprecipitation, and immunoelectron microscopy. It was synthesized as a 120-kDa precursor in infected cells and cleaved into 65-kDa and 55-kDa disulfide-linked subunits close to the time of virion release. The N-linked glycans on the cleaved, virion gB remained partially endoglycosidase H sensitive. The processing of MHV-68 gB therefore appears similar to that of Kaposi's sarcoma-associated herpesvirus gB and human cytomegalovirus gB.All herpesviruses express glycoproteins that attach virions to cells and fuse virion and cellular membranes (22). Such functions represent major therapeutic targets. The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, show only very limited infection of nonprimates, so murine gammaherpesvirus 68 (MHV-68), a close relative of Kaposi's sarcoma-associated herpesvirus (9, 31), has proved useful in identifying how gammaherpesvirus genes contribute to host colonization. MHV-68 has a broad cellular tropism that encompasses epithelial cells, B cells, macrophages, and dendritic cells (10,26,34). A major task in understanding how gammaherpesvirus glycoproteins influence pathogenesis is therefore to identify the glycoprotein functions of MHV-68.Glycoprotein B (gB) plays an important role in the entry of both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus virions into cells. Kaposi's sarcoma-associated herpesvirus gB binds to heparan sulfate (1) and ␣3␤1 integrin; the latter interaction makes a major contribution to infection (2). The major Epstein-Barr virus receptor binding proteins are gp350/220 (28) and gp42 (15), but Epstein-Barr virus gB is an essential protein (12) and high levels of gB in virions correlate with enhanced infectivity (19). MHV-68 lacks an obvious homologue of gp42, and its positional homologue of gp350/220, gp150, promotes virion release rather than binding (8). MHV-68 gB is therefore likely to play a major role in virion entry even though it lacks the RGD integrin binding motif (32) of Kaposi's sarcoma-associated herpesvirus gB.MHV-68 gB was originally described as a 110-kDa glycoprotein product of open reading frame 8 that was absent from virions and remained confined to the endoplasmic reticulum during lytic infection (24). A subsequent report identified gBderived peptides in an 88-kDa virion protein, with the discrepancy attributed to different methods of virion purification (7). Clearly, it is important in defining the molecular basis of MHV-68 tropism to determine whether gB is consistently found in virions, as it is in Kaposi's sarcoma-associated herpesvirus (1, 4) and bovine herpesvirus 4 (16). Our data demonstrate that gB is readily detectable in virions, mainly as 65-kDa and 55-kDa cleavage products of a full-length 120-kDa protein. Cleavage was associated with gB incorporation into virions. The MHV-68 gB is therefore a virion component that is processed very similarly to gB of Kaposi's sarcoma...

Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

“…The amounts of ORF45 protein in WT BAC and 33STOPϩHA-33 were similar, although the incorporation of HA-33 was much less than that of ORF33 in WT BAC, indicating that ORF33 could help viral particle maturation even at a low level of expression or incorporation. In WT BAC virions, glycoprotein B (envelope glycoprotein) was mainly detected as a 65-kDa protein (33). However, 33STOPϩHA-33 particles also contained another form of glycoprotein B with a smaller molecular mass that may be the 55-kDa form described by Lopes et al (33).…”

“…However, 33STOPϩHA-33 particles also contained another form of glycoprotein B with a smaller molecular mass that may be the 55-kDa form described by Lopes et al (33). Only the smaller form of glycoprotein B existed in 33STOP particles, which is consistent with our TEM result that no enveloped 33STOP viral particles was observed, since the 65-kDa protein is the abundant form of glycoprotein B in mature virions (33). Interestingly, a higher amount of actin protein was observed in 33STOP particles than in 33STOPϩHA-33 particles, whereas no actin protein was detected in WT BAC particles in Western blot analyses.…”

“…In WT BAC virions, glycoprotein B (envelope glycoprotein) was mainly detected as a 65-kDa protein (33). However, 33STOPϩHA-33 particles also contained another form of glycoprotein B with a smaller molecular mass that may be the 55-kDa form described by Lopes et al (33). Only the smaller form of glycoprotein B existed in 33STOP particles, which is consistent with our TEM result that no enveloped 33STOP viral particles was observed, since the 65-kDa protein is the abundant form of glycoprotein B in mature virions (33).…”

Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H

“…The gL -DEL and gL -DEL-STOP mutants also showed lower cell surface expression of other viral glycoproteins (Fig. 3A), particularly for gN (24) and gB (22), which are likely to reach the cell surface only in virions. The gB and gN content of gL Ϫ virions themselves was not reduced (Fig.…”

Murine Gammaherpesvirus 68 Open Reading Frame 11 Encodes a Nonessential Virion Component

“…The open arrowhead indicates the native ORF11 band; the filled arrowhead indicates the ORF11-CFP fusion band. (E) ORF11-CFP virions were treated as described for panel D but immunoblotted with either an anti-GFP rabbit serum (Molecular Probes) to detect the ORF11-CFP fusion protein or the anti-gB monoclonal antibody T7H9 (9). gB in virions is predominantly cleaved from a 120-kDa precursor, and T7H9 recognizes the 65-kDa C-terminal fragment (9).…”