Differential membrane binding of the human immunodeficiency virus type 1 matrix protein

“, 1997), together with the finding that deletions in the C terminus of MA (Zhou and Resh, 1996) or throughout the protein (Spearman et al.”

“The differential exposure of the myristic acid, through a process known as the myristyl switc”

“These include (i) the expression of Gag precursors and precursor variants in cells and the analyses of their membranebinding and particle-forming capabilities (90, 118, 215, 271, 310), (ii) in vitro synthesis of Gag related peptides and analysis of their interaction with liposomes (309), and (iii) purification of Gag…”

“In the case of human immunodeficiency virus type 1 (HIV-1), the Gag precursor, Pr55Gag, is cleaved by PR into four mature Gag proteins, i.e., p17 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC), and p6, and two spacer peptides, i.e., p2 and p1 (11, 55).”

“The myristate moiety is considered to be regulated by a mechanism termed a myristoyl switch (30, 57, 65, 72, 74, 75, 80, 84, 94).”

“The MA sequences include a carboxy-terminal α helix in the crystal structure [42,43], which juts away from the putative membrane-binding portion and has been shown to affect the conformation of the amino-terminal portion of MA by in vitro membrane binding [52].”

“viously (69) with the exception that the enhanced GFP (eGFP) coding sequence”

“This mutation abolishes Pr55 myristylation and severely impairs Gag membrane binding (4, 45, 61, 71).”

“Our finding that large deletions in MA increased particle yields may be explained by a recently proposed myristyl switch model of Gag membrane binding (Zhou and Resh, 1996).”

“p17 (MA) thus displays a significantly reduced affinity for membrane compared with Pr55 (42, 55).”

“This mechanism is known as the myristoyl switch (8, 9).”

“One explanation for the discrepant findings is that the addition of dihydrofolate reductase, green fluorescent protein, or bgalactosidase to the C terminus of MA in the studies of Zhou and Resh may have altered the structure of the globular head of MA.”

“Several retroviral Gag polyproteins have been proposed or shown to contain a cluster of basic amino acids in addition to N-terminal acylation [36,38].”

“It has been postulated that Gag processing induces conformational changes in the matrix (MA) domain of Gag (25, 44, 59).”

“…membrane targeting of HIV-1 Gag is regulated by a highly basic cluster of amino acids located between MA residues 17 and 31 (Freed et al., 1994; Hermida-Matsumoto and Resh, 2000; Ono et al., 2000a; Ono and Freed, 1999; Ono and Freed, 2004; Ono et al., 1997; Yuan et al., 1993; Zhou and Resh, 1996).”

“Such a model was originally proposed by Zhou and Resh to explain the relatively low affinity of mature MA for membranes, which may allow it to dissociate from the plasma membrane to participate in the nuclear import of the viral genome (53).”

“Previous work has demonstrated that HIV-1 Pr55 binds more efficiently to cellular or phospholipid vesicle membranes than MA (39, 40, 48).”

“05% saponin, and stained with a monoclonal antibody that recognizes p17 only in the context of mature virus (Ono et al., 2000; Zhou and Resh, 1996).”

“It has been observed that Pr55 binds membrane more tightly than MA itself (55, 64, 79), suggesting that cleavage of Pr55 may trigger conformational changes in MA which reduce its affinity for membrane.”

“Prior to membrane binding, the myristoyl moiety is sequestered in a hydrophobic patch on the MA domain (129), and its exposure may be regulated by PI(4,5)P2 binding (118) and multimerization of Gag molecules (129, 146).”

“Molecular biology experiments have demonstrated that MA plays essential roles in targeting PrGag proteins to membranes for assembly, and in incorporation of HIV SU/TM (gp120/gp41) envelope (Env) protein trimers into virus particles (Yu et al., 1992; Wang and Barklis, 1993; Wang et al., 1993; Facke et al., 1993; Dorfma…”

“In this manner, Nef may be removed from the viral membrane by a process analogous to the myristoyl switch mechanism proposed for regulating the association between the HIV-1 MA protein and the plasma membrane of the cell (41).”

“A possible explanation for the increased particle yields relative to those of the wild type is offered by the recently proposed myristyl switch model of Gag membrane targeting (35).”

“Other in vitro studies of the binding of myristoylated HIV-1 proteins to membranes used proteins derived from a rabbit reticulocyte in vitro transcription and translation system (15, 21, 50, 71, 72), which yields a radiochemically pure protein that is easily monitored.”

“According to this model, the myristate moiety is highly exposed in the context of Pr55 but is sequestered within p17(MA) following proteolytic cleavage of the precursor by PR (21, 48, 63).”

“p65-Gag-green fluorescent protein (GFP) chimeras have been previously described (36).”

“Several groups have now provided supporting data for the presence of the myristyl switch within MA (25, 26, 30, 43).”

“Mutating basic residues in the MA domain mistargets Gag, such that it associates with internal compartments rather than the plasma membrane [122,163–169].”

“Second, HIV-1 MA is reported to bind to membranes in vitro less efficiently than full-length Gag, suggesting a cooperative role for Gag multimerization in this process (Zhou and Resh, 1996; Sandefur et al., 1998).”

“In the WT context, Gag multimerization likely promotes the exposure of the myristate moiety (Hermida-Matsumoto and Resh, 1999;Perez-Caballero et al., 2004;Sandefur et al., 2000;Sandefur et al., 1998;Spearman et al., 1997;Tang et al., 2004;Zhou and Resh, 1996).”

“Hence, the membrane binding free energies for both monomeric HIV-1 Gag and EIAV Gag are weak (w−5 kcal/mol) but sufficient to direct a significant amount of protein to the plasma membrane surface, which is preferentially enriched in acidic phospholipids with respect to other intracellular membranes (Pomorski et al., 2…”

“Complex conformational switches can trigger changes in accessibility of the lipid moiety [10-13].”

“This steady-state distribution of p17MA was similar to that found for p17MA expressed exogenously in vitro and in vivo (30, 39).”

“The M domain is essential for mediating interactions of the Gag polyprotein with the inner leaflet of the plasma membrane, where both myristic acid and electrostatic interactions between basic residues within the N-terminal end of MA and negatively charged membrane phospholipids play a role (14, 46).”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Paillart and Gottlinger, 1999; Perez-Caballero et al., 2004; Saad et al., 2007; Spearman et al., 1997; Tang et al., 2004; Zhou and Resh, 1996), discussed further in Section 3.2.”

“, MA-CA) have suggested that Gag multimerization can enhance both membrane binding and DRM association (51, 74, 75, 78, 100).”

“It is possible that the structures of p17 (MA) and the MA domain of Pr55 are not equivalent; the observation that an anti-MA monoclonal antibody reacts with p17 (MA) but not with Pr55 (25, 39) suggests that this is the case.”

“…of the BIV-l matrix domain is regulated by proteolysis: In the context of a newly formed virion, the Gag polyprotein is cleaved by the viral protease, exposing an as-yet-unknown "signal" on the matrix domain that sequesters the myristate chain (Spearman et at., 1997; Zhou and Resh, 1996).”

“The interaction of the N terminus of Gag with membranes is facilitated by a myristoyl switch (31, 37, 45).”

“These effects confer concentration dependence on the HIV-1 Gag-membrane interaction and are consistent with the notion that multimerization triggers the “myristoyl switch” (18, 22, 25), thereby imparting a high degree of cooperativity on HIV-1 Gag trafficking to membranes.”

“, a conformational change during which the N-terminal myristoylation of Gag, initially sequestered in the MA, becomes exposed and is able to interact with membrane phospholipids (25, 40, 45, 47, 48, 53, 55, 61).”

“When cells expressing Gag-iYFP were immunostained with an anti-p17MA antibody, which only recognizes the mature, cleaved matrix domain of Gag [100,101], the YFP signal was observed to colocalize substantially with p17MA signal at the uropod (Figure 2A and Table 3).”

“Resh and colleagues proposed that Gag– membrane binding is regulated by a ‘‘myristyl switch’’ mechanism, whereby the myristate can shift between exposed and sequestered conformations.(39) Summers and coworkers used nuclear magnetic resonance (NMR) methods to confirm this hypothesis and to demonstrate that myristate ex…”

“74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107).”

“This concept agrees with the myristyl switch model in which a lesser extent of exposure of myristate acid is believed to be the reason for the weak association of MA with the plasma membrane in comparison with the results observed for Pr55 (27, 48, 55, 68).”

“amino-terminal acyl moiety and other motifs in MA (16, 57).”

“The subsequent cleavage event releases MA from CA-SP1 [126] leaving a mature MA associated with the membrane albeit with a considerably weaker affinity than MA within the context of Gag [73,77].”

“It has also been proposed that HIV-1 Gag protein in the cytosol may adapt a different conformation once it has bound to the membrane (31, 45).”

“model for the sequence of Gag(88-100) based on the C-terminal regions of other”

“In cells, myristoylation of Gag is a prerequisite for plasma membrane targeting (49–52), since mutation of the N-terminal glycine completely blocks membrane localization of Gag and the subsequent assembly of infectious virus particles (53).”

“Although many examples of lipidation-mediated compartment-specific localization of cytosolic proteins are found in the literatur”

“According to the hypothesis of a conformational myristoyl switch mechanism (Zhou and Resh, 1996; Spearman et al., 1997), the myristic acid has been proposed to be a key regulator of the Gag–membrane and MA–membrane interaction.”

“It was therefore unexpected when processed p17 (MA) was found to be present in postentry HIV-1 nucleoprotein complexes (7, 20, 25, 39); a conformational switch that results in the myristoyl residue becoming hidden from the surface of MA (53, 60), in conjunction with the phosphorylation of MA (6, 24, 25, 33), is though…”

“This anomaly in the defective particles might hinder the disassembly process, as has been postulated for the uncoating of the incoming particle during human immunodeficiency virus infection (32, 49).”

“Zhou and Resh (1996) have proposed that, when the myristate is exposed, it enables HIV Gag to bind todirects Gag to an intracellular assembly site.”

“This supports a pre-existing hypothesis first proposed by Zhou et al [31]: the existence of a “myristyl switch”, that is actually an entropic equilibrium between the [T] conformation that sequesters the myristate inside the protein, and the [R] conformation that promotes trimeristation and exposure of the myristate mo…”

“In vitro studies have established that Gag binds membranes more efficiently than the isolated MA protein (Zhou and Resh, 1996; Spearman et al., 1997; Hermida-Matsumoto and Resh, 1999; Ono and Freed, 1999; Paillart and Gottlinger, 1999; Bouamr et al., 2003).”

“Evidence supports a myristoyl switch model for HIV, in which the MA myristate is partially buried until PrGag oligomerization, when its hydrocarbon tail is exposed, fostering membrane binding and virus assembly (5, 15, 22, 47, 49, 54, 56, 61).”

“Indeed, a correlation between Gag multimerization and Gag-membrane binding has been proposed in a “myristyl switch” model (21, 42, 45, 57).”

“The myristoylated moiety is cotranslationally coupled to the p55 MA domain, and its exposure is regulated by binding to calmodulin: binding to calmodulin induces conformational changes within MA [93,94] which lead to p55 binding to lipid rafts [95-97], where it oligomerises [73,86,87,98], through the C-terminal domain…”

“Why is the M domain inactive in this context? It is possible that cleavage of MA from the remainder of Gag allows conformational rearrangements that disrupt the M domain and prevent plasma membrane binding (37, 50).”

“HIVgptmyr encodes a glycine-to-alanine substitution at residue 2 of MA, which abolishes the Pr55 myristylation signal and severely impairs Gag membrane binding (7, 47, 55, 72).”

“This result is consistent with the myristoyl switch model (16,17,23,24).”

“This difference in membrane binding is believed to be due to a myristoyl switch masking the hydrophobic properties of MA (41, 49).”