The HIV-1 assembly machine

Introduction HIV-1, like all retroviruses, is released by budding from the host cell's surface, and thereby acquires a plasma membrane-derived lipid envelope that provides a tight seal and shields the virus from the environment. Budding also offers a non-lytic pathway for virus egress, and thus has the advantage that the infected cell remains viable and can continue to produce progeny virions. HIV-1 particle assembly and budding are directed by the Gag polyprotein, the precursor for the internal structural components of the mature virion, which are matrix (MA), capsid (CA), nucleocapsid (NC), and p6. The Gag polyprotein coordinates the late stages of the replication cycle by bringing together all the building blocks of the virion, and is sufficient to organize the efficient assembly and release of virus-like particles even when expressed in the absence of other viral proteins. Initially, the polymerization of Gag polyproteins leads to the formation of an immature particle with a relatively stable spherical shell underneath the virion envelope. However, for the virion to become infectious, a maturation step is required that results in a suitably metastable capsid that can be disassembled within a target cell to release the viral genome. For maturation to occur, the Gag polyprotein must be cleaved by the viral protease, which triggers a large-scale rearrangement of the cleavage products within the assembled particle and a dramatic structural transition from a spherical to a conical capsid [1-4]. The structure of the Gag polyprotein and the precise nature of the interactions that enable it to assemble into a spherical protein shell capable of extruding through the plasma membrane remain unknown. However, substantial progress has been made in the recent past toward understanding the structure and function of each of the four major domains of the Gag polyprotein in virus morphogenesis. Matrix The N-terminal MA domain enables the Gag precursor to interact with the plasma membrane. The membrane targeting function of MA depends on the covalent attachment of myristic acid to an N-terminal glycine residue, which is absolutely essential for virus particle formation and HIV-1 replication [5,6]. However, since the membrane-binding energy contributed by the insertion of myristic acid into the lipid bilayer is small, additional determinants in MA are thought to contribute to the stable membrane association of Gag [7]. Indeed, the phenotypes of certain MA mutants indicate that the globular core of MA also has an important role in membrane binding [7-9]. The three-dimensional structure of HIV-1 MA reveals a globular 'head' formed by four α-helices and a C-terminal α-helix that projects away from the core domain [10-12]. The globular head forms trimers in all known crystal forms, and conserved basic residues cluster on the upper surface of the trimer, where they could potentially interact with acidic phospholipid head groups [12]. In support of this notion, the presence of a highly basic surface region is a common feature of retroviral MA proteins [13-15], and was recently shown to play a critical role in the plasma membrane localization of Rous sarcoma virus (RSV) Gag [16]. Interestingly, in the latter study, basic residues involved in membrane binding could be repositioned without affecting particle release [16], indicating that there is a certain degree of flexibility in the interaction of MA with anionic phospholipids. Accumulating evidence suggests a model in which the ability of HIV-1 MA to interact with membranes is regulated by a myristyl switch mechanism [17-20]. In this model, the myristic acid moiety would be sequestered by MA until a conformational change leads to its exposure and makes it available for membrane insertion. The model predicts that certain mutations should compromise the ability of MA to bury the myristyl group, which may explain why alterations within the α-helical core of MA often lead to substantial increases in membrane binding and, in some cases, clearly enhance viral particle yields [17,19,20]. Moreover, even deletions that remove the globular core of MA entirely can significantly increase HIV-1 particle production in cell culture [21]. Interestingly, deleting the globular core of MA also appears to facilitate assembly in vitro, because full-length HIV-1 Gag or a version lacking p6 formed only small spherical particles [22], whereas a Gag protein lacking both p6 and the globular core of MA formed particles that resembled authentic virus in size [23]. Taken together, these results support the view that MA can assume a conformation that interferes with assembly, and that a conformational switch is needed to trigger efficient Gag polymerization. If this switch regulates the availability of the myristyl group for membrane insertion, then disrupting the globular core of MA may cause increased Gag membrane binding because the myristyl moiety is now constitutively exposed. This model could also accommodate the observation that even conservative single amino acid substitutions near the N-terminus of MA can severely reduce Gag membrane binding and viral particle formation without affecting Gag myristylation [19,20,24]. The effects of these N-terminal mutations on Gag membrane binding resemble those seen in the absence of the myristyl acceptor site, indicating that they interfere with the exposure of the myristyl group. Remarkably, the defects in membrane binding, assembly, and virus replication can all be reversed by second-site mutations in the globular core of MA that increase Gag membrane binding [19,20], perhaps because they disrupt the ability of MA to sequester the myristyl group. A conformational switch that activates Gag membrane binding could, in principle, account for the selective targeting of Gag to the plasma membrane. However, recent results suggest that there is no direct relationship between Gag membrane binding and Gag targeting [25]. One et al. observed substitutions in MA where retargeted assembly to intracellular membranes did not always affect the extent of Gag membrane binding. Conversely, a mutation in the basic region of MA did not induce Gag re-targeting even though it increased the fraction of Gag that was membrane associated [25]. Interestingly, whereas large deletions in MA apparently lead to promiscuous assembly at the most abundant cellular membrane, the endoplasmic reticulum [26], single amino acid substitutions in MA can re-target HIV-1 assembly to the Golgi and/or Golgi-derived vesicles [25]. These observations raise the possibility that an association of Gag with Golgi-derived compartments prior to assembly at the plasma membrane may play a previously unsuspected role in the viral life cycle. Besides its key role in Gag membrane targeting, a second essential function of MA during assembly is to allow the incorporation of the HIV-1 envelope (Env) glycoprotein spikes. These spikes consist of an oligomeric complex formed by the surface glycoproteins and the transmembrane glycoproteins (TM). Although HIV-1 Env incorporation into assembling particles is often blocked by mutations in MA or in the cytoplasmic domain of TM [27-32], it is clear that a specific interaction between these two components is not always necessary. Indeed, several lines of evidence support the concept that MA is primarily required to passively accommodate the long cytoplasmic tail of HIV-1 TM into the particle. First, MA mutants that exhibit an absolute block in the incorporation of the HIV-1 Env complex readily accept heterologous viral Env glycoproteins with short cytoplasmic domains [30,33]. Second, the incorporation of the autologous HIV-1 Env complex into MA mutants can be fully rescued by second-site mutations that shorten the cytoplasmic tail of TM [30,33]. Third, at least in transient expression systems, the entire MA domain becomes dispensable for efficient Env incorporation if the cytoplasmic tail of HIV-1 TM is deleted [21]. Remarkably, if TM is truncated, efficient HIV-1 replication can be observed in the complete absence of MA in at least one human T-cell line [21]. On the other hand, new studies provide direct support for a stable interaction between TM and Gag in immature HIV-1 particles [34] and for a cell-type dependent requirement for the MA-TM interaction in HIV-1 Env incorporation [35,36]. Removing the TM cytoplasmic tail reduced Env incorporation more than 10-fold in the majority of human T-cell lines and in primary cells, where the truncation prevents HIV-1 replication [35]. Similarly, cell-type-specific defects in Env incorporation were seen in the presence of small deletions within a putative α-helical region of the TM cytoplasmic tail [36]. Moreover, the Env incorporation defect of one of these TM mutants could be reversed by a single amino acid change (V34I) in MA, supporting the view that an interaction between MA and TM drives Env incorporation in infected T cells [36]. Intriguingly, the V34I substitution was independently observed in a revertant derived from a virus that combines wild-type gag and env genes from different strains of HIV-1 (A. Borsetti, T. Dorfman, H. Göttlinger, unpublished observation). Although the chimeric virus replicates efficiently in primary cells, it fails to spread in most transformed T-cell lines unless the V34I substitution in MA is present. Taken together, these results indicate that the MA-Env interaction can substantially influence the cell tropism of HIV-1, and that cell-type specific factors play a crucial role in Env incorporation. It is conceivable that the role of MA in Env incorporation is linked to its specific membrane targeting function, because recent studies suggest that both Gag and Env associate with detergent-insoluble membrane domains known as lipid rafts [37,38]. Capsid CA, which directly follows MA in the context of the HIV-1 Gag polyprotein, forms the core of the mature virion. Structural studies indicate that CA has two distinct, largely α-helical domains that are connected through a flexible linker region [39-41]. Consistent with this model, cryo-electron microscopy of immature HIV-1 or Moloney murine leukemia virus reveals a radial arrangement of protein layers, two of which can be assigned to the N-terminal and C-terminal domains of CA [42-44]. The two CA domains have different roles in virus morphogenesis. The larger N-terminal CA domain is required for the formation of the cone-shaped core of the mature virion but is dispensable for the assembly of immature viral particles [45-48]. In contrast, the C-terminal domain is crucial for Gag polyprotein multimerization and HIV-1 particle assembly [41,46-49]. The N-terminal CA domain interacts specifically with the peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA) and mediates its uptake into HIV-1 virions at a CA : CyPA ratio of approximately 10 : 1 [50-52]. Pharmacological agents or CA mutations that disrupt the interaction with CyPA have no evident effects on HIV-1 particle assembly, Gag processing, or virion maturation [51-53]. However, viral infectivity is reduced, indicating that the incorporation of CyPA is functionally relevant [51,52]. Recently, the regulatory role of CyPA in HIV-1 infectivity was formally demonstrated in a variant of the Jurkat T-cell line that lacks CyPA [54]. In contrast to HIV-1, closely related primate immunodeficiency viruses such as HIV-2 and simian immunodeficiency virus SIVmac do not require CyPA. However, the transfer of HIV-1 CA, or of the CyPA-binding site within CA, to SIVmac can confer an HIV-1-like sensitivity to CyPA-binding compounds, which strongly suggests that CA is not only the interaction partner, but also the functional target of CyPA [55,56]. It has been proposed that CyPA promotes HIV-1 core disassembly following entry into the host cell by destabilizing CA interactions [57], but a new study shows that CyPA does not efficiently dissociate in vitro assembled CA cylinders [58]. Furthermore, recent image reconstructions of CA tubes suggest that the CyPA-binding loop is on the exterior surface of the viral core, and that isolated CyPA molecules can be modeled onto the surface without steric clash and are thus unlikely to promote disassembly [59]. Based on the observation that CyPA at the same molar ratio to CA as in the virion increases the efficiency of CA assembly in vitro, it was recently proposed that CyPA acts as a core assembly chaperone during virus maturation [58]. The C-terminal CA domain begins with the major homology region (MHR), a stretch of about 20 amino acids that is uniquely conserved among retroviral CA proteins [41]. The C-terminal CA domain dimerizes in solution with nearly the same efficiency as intact CA; however, the MHR does not contribute to the dimer interface [41]. In the context of the Gag precursor, the CA dimerization domain may extend into the adjacent p2 'spacer' peptide, which is predicted to form part of an α-helix that begins in CA [60]. Mutations that disrupt the putative α-helical region at the CA-p2 boundary lead to the assembly of large electron-dense patches or long tubular structures at the cell membrane, indicating that p2 is required for the induction of curvature [5,60-62]. Cleavage at the CA-p2 junction during virus maturation, which is expected to break the helix and to weaken CA-CA interactions, is required for the rearrangement of the spherical CA shell into a cone-shaped core [60,63]. In a recent study, the C-terminal CA-p2 domain was sufficient to direct efficient VLP formation when combined with a minimal membrane anchor, a heterologous protein-protein interaction domain, and a short peptide that promotes virus release [64]. This result points to a high degree of plasticity in the assembly process and confirms that the C-terminal CA domain is at the heart of the HIV-1 assembly machinery. In vitro, CA spontaneously assembles into long helical tubes and into cones that resemble mature viral cores [59,65-67]. However, the addition of as few as four MA residues to the N-terminus of CA prevented tube formation and instead resulted in the assembly of spherical structures more reminiscent of immature capsids [66,67]. Based on the three-dimensional structure of the N-terminal CA domain, it was proposed that cleavage at the MA-CA junction creates a new CA-CA interface essential for core assembly by allowing Asp51 of CA to form a salt bridge with the N-terminal CA proline residue [66]. Nevertheless, a new study shows that this salt bridge is not absolutely necessary for tube formation in vitro if additional domains of the Gag precursor are present [23]. A Gag precursor that lacked only a portion of MA and the C-terminal p6 domain assembled into either spheres or tubes and cones, depending on the pH of the reaction [23]. Interestingly, sphere formation was blocked by deleting the p2 spacer peptide that separates CA and NC, whereas short tubes and cones could still be assembled [23]. In agreement with the data obtained in the viral context [60], these in vitro results suggest that p2 functions as a molecular switch region that controls the transition from sphere to cone formation required for virus maturation. Ganser et al. used synthetic cores assembled from HIV-1 CA-p2-NC to test their model that retroviral cores are composed of closed hexagonal lattices [68]. In this model, the conical shape of HIV-1 cores is determined by the presence of 12 pentameric defects. In addition to and all of the cone by the model were in observed in synthetic core [68]. On the other hand, the cone of authentic HIV-1 cores isolated from virions the of the by the hexagonal model cryo-electron image reconstructions of synthetic CA tubes that these are composed of [59]. that the on the tube exterior are formed by the N-terminal domains of CA, whereas the C-terminal CA domains to at a tube that each to its [59]. The three-dimensional structures of different of tubes indicate flexibility in the of the C-terminal domains to the N-terminal This flexibility the of a model in which the HIV-1 core is made from CA as observed in the and 12 to the of the cone [59]. of this model is that the core should be for small such as for required for of the viral genome. 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