Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly

Abstract

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.

In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of Human Immunodeficiency Virus

“…Release of HIV-like particles still occurred, albeit at very low efficiency, if the entire matrix domain was replaced by a myristoylation signal [54]. Internal deletion mutants lacking most of the globular core of the matrix protein, on the other hand, yielded efficient assembly of spherical immature cores which remained in the cytoplasm [55] or budded into the endoplasmic reticulum [56]. Future experiments will address a potential influence of the matrix protein on formation or shape of in virro assembled particles.…”

HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

“…The HIV Gag gene is a major structural protein of the HIV virus. The Gag protein has been frequently used for HIV vaccine schemes [34]. The DNA sequence corresponding to a 24 amino acid region of the MPER plus a 15 amino acid linker was amplified by PCR and then cloned into the hexon HVR2 shuttle vector as previously described [16].…”

HIV-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly

“…A myristylation signal and a cluster of basic residues located in the MA amino-terminus are responsible for Gag-to-membrane binding and plasma membrane targeting. Gag membrane binding is required for efficient Gag multimerization and virus assembly (Chang et al, 2007;Lindwasser and Resh, 2001;Ono et al, 2000a,b;Spearman et al, 1997Spearman et al, , 1994Yuan et al, 1993). Blocking myristylation by Gly substitution or mutations in downstream basic residues can markedly impair Gag membrane binding or plasma membrane targeting, resulting in blocked virus particle production (Bryant and Ratner, 1990;Freed et al, 1994;Ono and Freed, 1999;Ono et al, 2000a,b;Yuan et al, 1993;Zhou et al, 1994).…”