Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus

Abstract

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55 Gag , to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55 Gag , a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.

Implications for lipids during replication of enveloped viruses

“…Most importantly, it is well known that viral proteins are myristoylated and/or palmitoylated (Resh, 1999;Maurer-Stroh and Eisenhaber, 2004). For instance retrovirus Gag and Nef proteins are typically myristoylated (Hizi et al, 1989;Shoji et al, 1989;Yu and Felsted, 1992;Ono and Freed, 1999;Campbell et al, 2001) while Env glycoproteins are palmitoylated (Resh, 1999). Palmitoylation has also been observed in HA and M2 proteins of influenza virus (Melkonian et al, 1999;Tatulian and Tamm, 2000;Thaa et al, 2009).…”

Retroviral mRNA nuclear export elements regulate protein function and virion assembly

“…Analyses of Gag interactions with membranes were performed similar to that described by Ono and Freed (1999). Briefly, transfected HeLa or 3T3 cells were harvested at B40 h, and postnuclear supernatants (PNSs) were generated by sonicating the cells and pelleting nuclei at 1000 g. PNSs were adjusted to 70% sucrose (w/v) in 2.5 ml, and overlaid with 6 ml of 65% (w/v) and 2.5 ml of 10% (w/v) sucrose in a 14 Â 89 mm tube (Beckman) to create a discontinuous gradient between the 10 and 65% sucrose fractions.…”

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.…”