COMMENTARY How HIV finds the door Matthew Scott Lalonde and Wesley I. Sundquist1 Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112-5650 he plasma membrane (PM) is nonspecifically with internal cellular high melting temperatures. Evidence that a physical barrier that must be membranes (Fig. 1B) (2). There is also HIV Gag associates with (or induces) rafts T breached when pathogens like evidence that MA and NC are held in includes colocalization with raft markers HIV enter and exit cells. In most close proximity within the soluble Gag and impairment of virion assembly upon cell types, HIV egress begins when the protein, possibly augmented by an RNA cholesterol depletion (3). MS-based lip- structural Gag protein binds and assem- bridge, and that this “foldback” confor- idomic analyses also support the raft hy- bles on the inner leaflet of the PM (Fig. 1). mation helps prevent Gag polymerization pothesis by showing that HIV membranes Gag assembly distends the membrane into prior to membrane binding (Fig. 1A) (2, 6, are enriched in raft lipids, including a spherical virion that is released by a 9, 10). cholesterol, glycosphingolipid GM3, membrane fission reaction catalyzed by sphingomyelins, plasmalogen-phosphati- the cellular endosomal sorting complexes HIV Gag can discriminate dylethanolamine, and saturated fatty acids required for transport (ESCRT) machin- (18, 19). These studies also confirmed the ery (1). HIV must therefore target the PM between membranes expected enrichment of PS and PtdIns selectively to avoid futile release into in- (4,5)P2 in the viral membrane. tracellular compartments. Previous studies with different These observations raise the question of have revealed important aspects of the how the virus localizes to raft-like sites on targeting mechanism (2, 3), but the path- cholesterol and fatty the PM. Dick et al. begin to address this way is not yet fully understood. A study question by quantifying Myr-Gag binding in PNAS by Dick et al. takes us a step acid compositions. to synthetic liposomes in which PS and closer to that goal by showing that HIV cholesterol concentrations are varied sys- Gag can discriminate between membranes tematically (4). They also test the effects with different cholesterol and fatty acid When Gag molecules have reached the of changing the length and saturation of compositions, thereby helping to explain PM, they must bind and initiate virion the fatty acid acyl chains on PS and on the how viral assembly can be targeted to lipid assembly. The PM contains unusually high neutral lipid phosphatidylcholine (PC). rafts on the PM (4). concentrations of cholesterol, and its cy- Myr-Gag molecules were translated and fl HIV Gag is initially translated as a toplasmic lea et is enriched in acidic myristoylated in vitro, and membrane multidomain polyprotein that is ultimately phospholipids, particularly phosphati- binding was assessed by using a liposome cleaved by the viral protease to produce six dylserine (PS) and phosphatidylinositol flotation assay. As expected, Myr-Gag P separate proteins and peptides, termed 4,5-bisphosphate [PtdIns(4,5) 2] (11). binding generally increased with in- MA, CA, SP1, NC, SP2, and p6 (Fig. 1A). HIV Gag uses each of these lipid cues to creasing PS concentrations. The authors Gag cleavage is required for viral matu- recognize the PM. The Gag MA domain is also discover that cholesterol invariably ration and infectivity, but the unprocessed highly basic and therefore binds prefer- enhanced Myr-Gag membrane binding, Gag polypeptide contains all the infor- entially to acidic membranes like the PS- consistent with the lipid raft hypothesis. P mation necessary to assemble and bud rich PM (2, 4). In addition, PtdIns(4,5) 2 Beyond the clear enhancing effects of from cells. The different regions of Gag also performs a more specific role in HIV PS and cholesterol, the Myr-Gag binding work together during virus assembly and targeting (12) because the phosphoinosi- patterns were unexpectedly complex. budding, performing different primary tide, glycerol, and 2′-fatty acid groups can Binding affinities often varied significantly functions: MA binds membranes and has bind within MA and allosterically release when the PS and PC fatty acid chains an N-terminal myristate (Myr) fatty acid the sequestered Myr to insert into the PM were altered, as did the degree to which fl B P modification (2, 3), CA/SP1 forms a hex- inner lea et (Fig. 1 ) (13). PtdIns(4,5) 2 binding was affected by changing PS and agonal lattice that organizes the spherical binding may also competitively release cholesterol concentrations. These dra- virion (5), NC contains basic residues and MA-bound RNA to interact exclusively matic variations provide strong evidence zinc finger domains that capture the viral with the NC domain, favoring Gag as- that Myr-Gag can sense lipid composition RNA genome and serve as an assembly sembly and increasing membrane binding and structure. It will now be important to scaffold (6), and p6 contains epitopes that avidity (2, 6, 9, 10). test the influence of each lipid property bind and recruit host ESCRT proteins (1). Despite their elegance, these confor- independently (e.g., fatty acid chain In the fully assembled virion, Gag mole- mational switching models do not fully length, saturation, and lipid packing) be- cules align lengthwise, with their MA do- explain Gag membrane targeting, particu- cause the mechanistic bases for the dif- mains against the viral membrane and larly for retroviral Gag proteins that lack ferences in Myr-Gag binding are not yet NC–RNA complexes at the virion interior. myristates or PtdIns(4,5)P2 binding activi- easily discernible. For example, choles- Gag is translated in the cytoplasm, but ties (14–16). Indeed, several reports argue terol is known to condense and order acyl does not form large assemblies until it that even HIV Myr-Gag does not exhibit chains, so the authors tested the hypothe- reaches the PM (7). Hence, soluble Gag strong binding specificity for PtdIns(4,5)P2 sis that Myr-Gag might simply be sensing subunits must avoid assembling prema- over other acidic phospholipids (14, 17). cholesterol-induced membrane ordering. turely or binding irreversibly to internal Moreover, HIV does not bud promis- membranes as they traverse the cytoplasm. cuously from the PM, but rather exits from Gag sequesters the terminal Myr within sites that have the characteristics of lipid Author contributions: M.S.L. and W.I.S. wrote the paper. a hydrophobic pocket of MA (8), and rafts (3). Lipid rafts are thick, immiscible, The authors declare no conflict of interest. mutations that destabilize this “myristoyl ordered lipid patches that are enriched See companion article on page 18761. ” in conformation enhance membrane in cholesterol, sphingolipids, and other 1To whom correspondence should be addressed. E-mail: binding, but cause Gag to associate saturated lipids that pack tightly and with [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1215940109 PNAS | November 13, 2012 | vol. 109 | no. 46 | 18631–18632 Downloaded by guest on October 2, 2021 Fig. 1. Stages and molecular interactions of HIV Myr-Gag during virus assembly and budding. (A) Schematic illustrations show the domain structure of the Gag polyprotein and the different stages of Myr-Gag assembly and budding from the PM. (B) Expanded molecular models illustrate how PtdIns(4,5)P2 binding to MA can induce extrusion of the sequestered myristoyl group. The figure also highlights the importance of PS and cholesterol for Myr-Gag membrane binding. For simplicity, the lipid bilayer is shown composed of only dioleoylphosphatidylcholines, and lipid ordered and lipid disordered phases are not depicted. However, when cholesterol levels were leukemia virus and the acetylated Gag formation (which shrinks the circumfer- held constant and membrane order was protein of Rous sarcoma virus. ence of the interface between ordered and increased incrementally by adding the In addition to helping target viral bud- disordered phases) or membrane fission high-melting temperature lipid dis- ding to the PM, lipid raft localization (which eliminates the interface) (21). tearoylphosphatidylcholine, Myr-Gag may also enhance retroviral replication These forces may therefore help to explain binding did not correlate well with the by stimulating Gag oligomerization (20), why cholesterol is required for efficient degree of order. Hence, the physical increasing incorporation of the raft-asso- retroviral budding and entry (3). membrane property (or properties) being ciated Env complex (3), or helping to In summary, HIV Gag encodes a re- sensed remain uncertain, but it is possible promote cell-to-cell transfer of virus at markable number of different activities that Myr-Gag binds cholesterol directly or raft-like virological synapses (3). Raft tar- that ensure that the virus assembles at the that cholesterol and certain combinations geting may also provide the virus with right time and place, incorporates the viral of PS and PC fatty acid chains induce a membrane whose composition favors the Env and RNA components, recruits the more favorable clustering/exposure of fission and fusion reactions required dur- cellular ESCRT machinery, and has the acidic lipids. Regardless of the underlying ing the ensuing stages of virus budding and lipids it needs to bud from and enter cells cause, the membrane recognition proper- entry. Mismatches in the junctions be- efficiently. We now know that HIV ties of HIV Myr-Gag appear to be con- tween the ordered raft lipid phase and Myr-Gag binds preferentially to choles- served in other retroviruses because disordered bulk PM lipid phase are terol-rich membranes, and next need to similar binding patterns are observed for thought to create high-energy tension lines learn how this pattern recognition the myristoylated Gag protein of murine that can be minimized by membrane de- is achieved. 1.