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

Abstract

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha-or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells.

Bovine Herpesvirus Type 4 Glycoprotein L Is Nonessential for Infectivity but Triggers Virion Endocytosis during Entry

“…The gH/gL heterodimer has been shown to bind glycosaminoglycans (GAGs) in both KSHV (25) and . This translated to reduced binding of gL-deficient MuHV-4 virions on some cell types (23). In contrast, we did not observe any binding deficit for the BoHV-4 gL STOP mutant in the tested cell lines (Fig.…”

“…However, we recently showed that gL was not essential for murid herpesvirus 4 (MuHV-4), another rhadinovirus. MuHV-4 lacking gL both incorporates gH into virions and remains infectious, although it shows some attenuation relative to the wild type (WT) (23). This result is quite surprising compared to results for all alpha-and betaherpesviruses tested to date, in which gL proved to be indispensable (7,13,27,31,46).…”

Deletion of Murid Herpesvirus 4 ORF63 Affects the Trafficking of Incoming Capsids toward the Nucleus

“…Virus stocks were titrated by plaque assay on BHK-21 cells (34). Cell monolayers were incubated with virus (2 h at 37°C), overlaid with 0.6% carboxymethylcellulose, and 4 days later fixed and stained for plaque counting.…”

“…After 2 h of adsorption, the cells were washed then overlaid with DMEM containing 10% FCS. Supernatants of infected cultures and infected cells were harvested at successive intervals, and the amount of infectious virus was determined by plaque assay on BHK-21 cells (34). When stated, growth curves were performed in the presence of the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, 20 M; Promega) or of the NLRP3 inflammasome inhibitor glyburide (25 g/ml; Sigma).…”

“…Infectious virus in lungs was measured by homogenizing them in 6 ml of complete medium prior to plaque assay. Latent virus in ex vivo tissues was measured by infectious-center assay (34): spleen suspensions were cocultured with BHK-21 cells and then fixed and stained for plaque counting after 5 days. Preformed infectious virus titers in lymphoid tissue, as measured by plaque assay of freezethawed cells, were always Ͻ1% of the infectious-center assay titers, so the latter essentially measured reactivable latent virus.…”