Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.The assembly and budding of retroviruses involve a series of regulated steps, driven primarily by the viral Gag protein (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and budding occur predominantly at the plasma membrane. The HIV-1 Gag protein is expressed as a 55-kDa polyprotein, comprising four major domains and two spacer peptides (SPs). The major domains are matrix (MA), capsid (CA), nucleocapsid (NC), and p6; the spacer peptides are known as SP1 and SP2 and are located between CA-NC and NC-p6, respectively. Assembly and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral particles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3).In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 Env is synthesized as a 160-kDa precursor that traffics to the plasma membrane via the Golgi apparatus, where it is processed to form the surface glycoprotein gp120 and the transmembrane glycoprotein gp41 (reviewed in ref. 4). The processed Env glycoproteins remain noncovalently associated as a heterodimer; the Env spike is a homotrimer of these dimers (5, 6). On the surface of the viral particle, gp120 binds the viral receptor CD4 and the chemokine coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and coreceptor triggers structural changes in gp41 that lead to fusion of the viral and target cell membranes. The fusion activity of gp41 is conferred by the ecto- and transmembrane domains of the protein (7). A third domain, the cytoplasmic tail (CT), is dispensable for fusion but plays important roles in Env trafficking and cell signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a very long CT composed of ∼150 amino acids. In contrast, most other retroviruses encode Env CTs that are ∼25–35 amino acids in length (4). The reasons for the greater length of lentivirus Env CTs are not fully understood. For HIV-1, the CT is required for Env incorporation into particles in physiologically relevant cell types, such as peripheral blood mononuclear cells, monocyte-derived macrophages, and most T-cell lines (8, 9). Recent work has shown that the CT mediates an interaction with Rab11 family-interacting protein 1c (FIP1c), which in turn interacts with Rab14 and appears to direct trafficking of HIV-1 Env to sites of assembly; it is likely that interactions with FIP1c contribute to the observed requirement for the CT in Env incorporation (10, 11). It is, however, unclear why lentiviral CTs are so large, because far smaller Env CTs also contain essential functional trafficking and signaling motifs (12).A consequence of the large lentiviral CT is the potential for, or inevitability of, interactions with the MA domain of Gag during particle assembly. The Gag protein forms a hexameric lattice, driven primarily by CA–CA interactions. Whereas the structure of CA in mature particles has been studied extensively, with many high-resolution structures now available (13–16), and recently progress has been made in determining the structure of the immature Gag lattice (17), the organization of MA in particles has proven difficult to address directly, with no long-range order discernable for the MA shell. The structure of HIV-1 MA has been solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either approach; however, crystallography suggests a trimeric arrangement for both HIV-1 and simian immunodeficiency virus (SIV) MA proteins (19, 20), whereas NMR reveals only structures for the monomer, with no evidence for higher-order interactions (18). A third approach visualized 2D lattices of MA or MA-CA, using myristylated proteins on a synthetic lipid bilayer with a composition intended to mimic that of the plasma membrane at sites of assembly (21). Under these conditions, MA was seen to arrange as hexamers of trimers, although the low resolution of this approach precluded more-detailed structural analysis. A hexamer-of-trimers arrangement for MA would be compatible with the most recently suggested model for the immature CA lattice, which proposes a hexamer-of-trimers arrangement for the CA amino-terminal domain (CA-NTD), which lies immediately below MA (17).The available structures of MA can be used to place the data acquired through molecular and genetic approaches into context. Mutations have been identified in MA that prevent the incorporation of Env into particles (22–26). The majority of these mutations map to the tips of the MA trimer (27), a region of the protein that lies around the central aperture of the hexamer of trimers. These findings implicate this central aperture as the site of Env incorporation in the particle. This idea is further supported by the observation that, in cell lines permissive for packaging of CT-truncated Env, removal of the CT relieves the inhibition of Env incorporation imposed by the MA mutations (22, 25, 28). The ability to rescue Env incorporation by removing the CT suggests that steric hindrance of Env incorporation may be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data showing that a mutation at the trimer interface was able to rescue the Env incorporation defects imposed by several MA mutations and a deletion in the Env CT (24). These data suggest that the MA trimer interface regulates the ability to rescue mutants that are defective for Env incorporation.In this study, we sought to develop a system that would allow us to directly determine whether MA forms trimers in virions and, if so, whether MA trimerization plays a role in Env incorporation. By using a combination of biochemical, genetic, and virological approaches, we demonstrate the presence of MA trimers in replication-competent HIV-1 particles, and show a strong correlation between loss of MA trimerization and impaired Env incorporation. These MA trimerization-defective mutants could be rescued by removal of the long Env CT, demonstrating that the requirement for MA trimerization in Env incorporation is linked to the presence of the long gp41 CT. This report both demonstrates the existence of MA trimers in infectious HIV-1 particles and establishes the importance of this structure for Env incorporation.