Point Mutations in the HIV-1 Matrix Protein Turn Off the Myristyl Switch

Abstract

During the late phase of HIV-1 replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ) and Gag's N-terminally-myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P 2 binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P 2 is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P 2 -or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr (s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5) P 2 binding. This mutant binds PI(4,5)P 2 with two-fold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding. KeywordsHuman immunodeficiency virus type-1 (HIV-1); Gag; myristyl (myr); matrix (MA); phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ); analytical ultracentrifugation (AU); nuclear magnetic resonance (NMR)

NMR, Biophysical, and Biochemical Studies Reveal the Minimal Calmodulin Binding Domain of the HIV-1 Matrix Protein

“…Furthermore, Leu-8 and Ser-9 have been shown to be critical for the function of the myristyl switch mechanism and membrane binding (78 -81). Subsequent structural studies revealed that substitution of Leu-8 and Ser-9 shuts off the myristyl switch mechanism, which severely inhibit Gag targeting to the PM (50). The finding that Leu-8 and Ser-9 act as stabilizing residues of the CaM-MA complex may suggest a dual role in Gag assembly.…”

“…The N-terminal region of MA (residues 2-47) is critical for diverse Gag functions including Gag-membrane interactions, regulation of the myr switch mechanism, and binding to several cellular constituents implicated in Gag trafficking and/or gp160 incorporation (7,17,18,23,24,28,30,50,57,(71)(72)(73). Several residues in helix I of MA (Leu-8, Ser-9, Leu-13, Trp-16, Glu-17, and Lys-18) are important for gp160 incorporation (74 -76), which led to the suggestion that the MA domain of Gag interacts directly with the gp160 protein via helix I (74,77).…”

“…Protein Expression and Purification-The unmyristoylated MA (myr(Ϫ)MA) protein was expressed and purified as described (49,50). CaM samples have been prepared as described with minor modifications (51,52).…”

Une nouvelle vision de l’assemblage du VIH-1

“…Deux structures alternatives de MA du VIH-1 ont été publiées, dans lesquelles le myristate est soit enfoui dans la structure protéique, soit exposé à sa surface. Son exposition serait déclenchée par l'oligomérisation de Gag et son interaction avec le PI(4,5)P2 [10], et inhibée par sa maturation protéolytique [11], suggérant que la stabilité de l'association de Gag avec les membranes pourrait être renforcée transitoirement au moment de l'assemblage et du bourgeonnement, et revenir au niveau basal après maturation de Gag par la protéase virale. Ces données suggèrent un modèle dans lequel l'assemblage de Gag est guidé par une double plateforme constituée de l'ARNg, dont l'interaction spécifique avec NC permet la nucléation de l'assemblage et sa propre incorporation Lors des phases tardives de la réplication virale, les molécules de Gag sont synthétisées au niveau des polysomes, sans doute dans le cytoplasme et se multimérisent sur l'ARN génomique viral (ARNg) grâce au domaine nucléocapside (NC) (1), formant ainsi des complexes ribonucléoprotéiques (RNP) viraux qui s'ancrent aux membranes de la cellule infectée.…”

“…Le caractère chevauchant de beaucoup de ces signaux est à ce propos significatif. Ainsi, le domaine amino-terminal de MA est responsable de multiples interactions de Gag : avec les phospholipides membranaires impliqués dans l'arrimage aux bicouches lipidiques, le PI(4,5)P2 (assurant un ciblage à la membrane plasmique [10]), AP-3 (le ciblant au contraire vers les MVB [20]), et TIP47, impliqué dans le recrutement de Env [27]. On peut difficilement envisager que ces interactions soient simultanées.…”