Herpes 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.

Membrane fusion during the entry of herpesviruses is carried out by the viral fusogen gB that is activated by its partner protein gH in some manner. The fusogenic activity of gB is controlled by its cytoplasmic (or intraviral) domain (gBCTD) and, according to the current model, the gBCTD is a trimeric, inhibitory clamp that restrains gB in the prefusion conformation. But how the gBCTD clamp is released by gH is unclear. Here, we identified two new regulatory elements within gB and gH from the prototypical herpes simplex virus 1: a surface pocket within the gBCTD and residue V831 within the gH cytoplasmic tail. Mutagenesis and structural modeling suggest that gH V831 interacts with the gB pocket. The gB pocket is located above the interface between adjacent protomers, and we hypothesize that insertion of the gH V831 wedge into the pocket serves to push the protomers apart, which releases the inhibitory clamp. In this manner, gH activates the fusogenic activity of gB. Both gB and gH are conserved across all herpesviruses, and this activation mechanism could be used by other gB homologs. Our proposed mechanism emphasizes a central role for the cytoplasmic regions in regulating the activity of a viral fusogen.

Membrane fusion during the entry of herpesviruses is carried out by the viral fusogen gB that is activated by its partner protein gH in some manner. Unusually, the fusogenic activity of gB is controlled by its cytoplasmic (or intraviral) domain (gB CTD ) and, according to the current model, the gB CTD is a trimeric, inhibitory clamp that restrains gB in the prefusion conformation. But how the gB CTD clamp is released by gH is unclear. Here, we identified two new regulatory elements within gB and gH from the prototypical herpes simplex virus 1: a surface pocket within the gB CTD and residue V831 within the gH cytoplasmic tail. Mutagenesis and structural modeling suggest that gH V831 interacts with the gB pocket. The gB pocket is located above the “fault line” between adjacent protomers, and we hypothesize that insertion of the gH V831 wedge into the pocket serves to push the protomers apart, which releases the inhibitory clamp. In this manner, gH activates the fusogenic activity of gB. Both gB and gH are conserved across all herpesviruses, and this activation mechanism could be used by other gB homologs. Our proposed mechanism emphasizes a central role for the cytoplasmic regions in regulating the activity of a viral fusogen.

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.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.