Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication

Natalia Saiz-Ros,Charles Dixon,Eric Schirmer,Martin Goldberg,Marion Mcelwee,Selene Swanson,Lior Pytowski,Dario Zamora,Rafal Czapiewski,Swetha Vijayakrishnan,David Kelly,Laurence Florens,Ilaria Epifano,Sheila Graham,Andrew Stevenson,Li Dong,Christine Richardson

doi:10.3390/cells8020120

Abstract

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Highlights

Herpesvirus genome replication and encapsidation occurs in the host cell nucleus
Their residence in both membranes could be brief due to recycling back to the inner nuclear membrane (INM), we reasoned that a proteomics analysis of nuclear envelope (NE) and ER membrane fractions may provide some clues as to the types of cellular proteins that might take part in HSV nuclear egress
During Herpes simplex virus type-1 (HSV-1) infection activated protein, kinase C is recruited to the nuclear membrane, where it phosphorylates and remodels the nuclear lamina to facilitate primary envelopment [57]. p32 is a cellular protein that can interact with a number of herpesvirus proteins and in HSV-1 infection, it is recruited to the nucleus/nuclear rim by ICP27 and ICP34.5 [23,58], but may play a role in nuclear egress via interaction with tegument protein UL47 [24]

Summary

Introduction

Herpesvirus genome replication and encapsidation occurs in the host cell nucleus. The capsids at~120 nm cannot pass through nuclear pore complexes [1], and so must traverse the nuclear envelope (NE) before virion maturation in the cytoplasm [2]. Capsids obtain a primary envelope from the inner nuclear membrane (INM), resulting in access to the NE lumen They exit the lumen by fusing with the outer nuclear membrane (ONM), leaving their primary envelope behind [3,4,5]. PUL31 and pUL34 are key viral proteins for HSV-1 nuclear egress They recruit host kinases to locally dissolve the intermediate filament nuclear lamin polymer to allow nucleocapsid access to the INM [6,7,8,9] and it has been suggested that they may promote vesiculation in vitro and in vivo [10,11]. Though its specific function is not clear, HSV-1 pUS3 contributes to nuclear egress as primary enveloped particles accumulate in the NE lumen when it is depleted [12,13,14]. This is observed for a combined knockdown of HSV-1 glycoproteins gB and gH that are components of the final envelope [15,16]

Methods

Results

Conclusion