Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release

Himanshu Garg,Sherimay D. Ablan,Eric O. Freed,Anjali Joshi

doi:10.1074/jbc.m111.241521

Abstract

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.

Highlights

Retrovirus assembly involves the participation of three key viral components: the Gag polyprotein precursor, the envelope (Env) glycoproteins, and the viral genomic RNA
2 The abbreviations used are: PM, plasma membrane; BFA, brefeldin A; CA, capsid; Carboxyfluorescein succinimidyl ester (CFSE), carboxyfluorescein succinimidyl ester; Cytochalasin D (CytoD), cytochalasin D; DN, dominant negative; Equine infectious anemia virus (EIAV), equine infectious anemia virus; ESCRT, endosomal sorting complex required for transport; MA, matrix; MLV, murine leukemia virus; multivesicular bodies (MVBs), multivesicular body; NC, nucleocapsid; NSF, N-ethylmaleimide-sensitive factor; PI[4,5]P2, phosphatidylinositol [4,5]-bisphosphate; SNAP, ␣-soluble NSF attachment protein; t, target membrane-associated; v, vesicle-associated
HeLa cells were transfected with siRNAs specific for NSF or SNAP23, and the effects on human immunodeficiency virus type 1 (HIV-1) particle production were determined by metabolic radiolabeling and immunoprecipitation analysis

Summary

Introduction

Retrovirus assembly involves the participation of three key viral components: the Gag polyprotein precursor, the envelope (Env) glycoproteins, and the viral genomic RNA. These components are typically transported to the plasma membrane (PM) where they assemble into nascent virus particles [1, 2]. In addition to the requisite viral Gag polyprotein precursor, assembly and release of retroviral virus-like particles are promoted by a variety of host factors. A dominant negative (DN) disruption of SNARE activity occurs on mutation of the ATP binding site (E329Q) of NSF that results in failure of NSF to disassemble the SNAP-SNARE complex due to a significant decrease in NSF ATPase activity (46 – 48)

Methods

Results

Conclusion