Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion

Abstract

Herpesviruses – ubiquitous pathogens that cause persistent infections – have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact prior to receptor engagement and that binding of gD to its receptor causes a “cascade” of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form prior to fusion, remain stable throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised “conformational cascade” model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex prior to fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems.IMPORTANCEHerpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form stable, receptor-independent complexes. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.

A surface pocket in the cytoplasmic domain of the herpes simplex virus fusogen gB controls membrane fusion

“…First, we do not yet have direct evidence of an interaction between the gB CTD pocket and the gH CT wedge. Recently, we reported that gH/gL and gB interact through multiple domains, independently of gD [ 62 ]. Disrupting the interactions within the cytoplasmic regions did not reduce overall gH-gB interaction, presumably due to the remaining interactions between the ectodomains and the TMDs.…”

“…Second, it remains to be elucidated what changes occur in gH that cause gH V831 to perform our proposed wedging action in the gB CTD pocket. We hypothesize that pre-existing interactions between the gB CTD and the gH CT [ 62 ] position the gH V831 wedge near the gB CTD pocket. Binding of gD to one of its cognate receptors activates gH/gL, causing it to undergo a conformational change that would push the wedge deeper into the gB CTD pocket, triggering the fusogenic refolding of gB.…”