Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids

Abstract

Retroviral Gag proteins are targeted to the plasma membrane, where they play the central role in virion formation. Several studies have suggested that the membrane-binding signal is contained within the amino-terminal matrix sequence; however, the precise location has never been determined for the Gag protein of any retrovirus. In this report, we show that the first 31 residues of human immunodeficiency virus type 1 Gag protein can function independently as a membrane-targeting domain when fused to heterologous proteins. A bipartite membrane-targeting motif was identified, consisting of the myristylated N-terminal 14 amino acids and a highly basic region that binds acidic phospholipids. Replacement of the N-terminal membrane-targeting domain of pp6o-src with that of human immunodeficiency virus type 1 Gag elicits efficient membrane binding and a transforming phenotype. Removal of myristate or the basic region results in decreased membrane binding of Gag-Src chimeras in vitro and impaired virion formation by Pr55rag in vivo. We propose that the N-terminal Gag sequence functions as a targeting signal to direct interaction with acidic phospholipids on the cytoplasmic leaflet of the plasma membrane.

Implications for lipids during replication of enveloped viruses

“…The complementary charge state of Gag MA and inner plasma membrane surface intuitively suggests an electrostatic mechanism by which Gag interacts with the membrane. This early hypothesis was supported by liposome pull-down assays showing that Gag can be enriched by liposomes containing PS (Zhou et al, 1994). A link between phosphoinositides and retrovirus assembly was provided by the observation that inositol polyphosphate molecules are able to promote cell free assembly of virus like particles from recombinant HIV-1 Gag molecules (Campbell et al, 2001).…”

“…HCMV (Machesky et al, 2008) Fatty acids Genome replication is stimulated by saturated or monounsaturated fatty acids. FAS is highly enriched in the supernatant of infected cells HCV (Su et al, 2002) Assembly/budding Phosphoinositides Gag binds specifically to PI(4,5)P2 causing a myristic switch in MA which increases membrane partitioning of Gag HIV (Zhou et al, 1994;Ono et al, 2004;Freed, 2006) Sterols It is also possible that PI(4,5)P2 interactions with cellular proteins contribute to membrane curvature during virus assembly and budding HIV (Chan et al, 2008) Surface exposed polybasic domains of similar matrix proteins also use phosphoinositides or other anionic phospholipids for "lipid-raft" targeting/induction of membrane curvature Influenza virus (Ruigrok et al, 2000;Thaa et al, 2009), EBOV (Timmins et al, 2004), VSV (Gaudier et al, 2002), RSV a (Money et al, 2009) Virus assembly occurs on lipid droplets HCV (Miyanari et al, 2007)…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…In immature viral particles, proteolytic processing generates several distinct products, including MA (matrix protein), CA (capsid protein), and NC (nucleocapsid protein), thus producing mature infectious particles. The membrane-binding domain of the MA protein directs the association of Gag with membrane, typically through a bipartite motif consisting of a covalently attached myristic acid moiety and a highly basic domain (Yuan et al, 1993;Zhou et al, 1994). There is considerable evidence that the N-terminal myristyl group of MA protein plays a role in regulating membrane binding (Tang et al, 2004).…”

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

“…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). The assembly domain involved in intermolecular interactions during Gag multimerization is largely located in the C-terminal domain of CA and the downstream NC region, and most mutations introduced into these regions can significantly impair virus particle assembly.…”