Structure of a monomeric mutant of the HIV-1 capsid protein

Retroviruses typically consist of a central capsid core particle encapsidating two copies of RNA and the viral enzymes. The capsids are composed of about 1500 copies of a capsid protein (CA) that is initially part of a Gag polyprotein synthesized in the infected host cell (1,2). The retroviral capsid proteins are typically ~24 to ~27 kDa in size, and are highly α-helical. The Gag proteins capture the viral RNA, assemble either in the cytosol (B and D-type retroviruses) or at the cell membrane (C-type, HTLV/BLV and lentiviruses) and bud into the enveloped immature virus particles (2). Gag is then proteolytically cleaved by the viral protease into the major structural proteins of the virus (2,3), followed by a maturation process in which the capsid proteins condense to form the mature capsid of the virus with a distinct shape characteristic of the genus (3). The HIV-1 capsid is a conical shaped fullerene structure (4). The capsid protein (CA) of the HIV-1 virus plays a significant role in the early stages of the viral life cycle, controlling the virion size, morphology and Gag assembly (5–7). Electron cryotomography images of the immature virions have shown that along with the spacer SP1, CA domains also play an important role in the formation of the hexameric Gag lattice (5). Most importantly, intermolcular CTD-CTD interactions appear to be important in the assembly of the hexameric Gag lattice (5,7). Electron microscopy studies show that the mature capsid of HIV-1 is as fullerene cone, with its surface composed primarily of hexameric CA rings, with twelve pentameric rings of CA that allow the cone to close at both ends (4). The surface of the mature capsid of HIV-1 is composed of a hexameric (and pentameric) rings of the N-terminal domains (NTD) stabilized by NTD-NTD interactions, with each ring linked to neighboring hexamers through the inter-hexamer dimerization of the C-terminal domains (CTD). Additional intermolecular NTD-CTD and CTD-CTD interactions further stabilize the mature capsid surface lattice (1,3,8,9). Thus, because of the critical role of CA in the assembly of the immature particles and mature capsids, recently there has been a rather significant interest in the CA protein as an antiviral therapeutic target to design inhibitors of early and late stage events in the HIV-1 virus replication cycle (1,4,10–15). Thus the availability of the structure of the full-length HIV-1 CA monomer would be of critical importance for efforts in the structure-based design of inhibitors. Such a monomeric structure will also facilitate a structural biological characterization of the interactions of the HIV-1 capsid protein with host cell proteins exploited by the HIV-1 virus in its replication cycle, such as cyclophilin A and lysysl-tRNA synthetase. However, HIV-1 wild-type full-length CA monomer protein has defied structural determinations by X-ray crystallography and NMR spectroscopy because of the high degree of flexibility of the inter-domain linker which made it difficult to crystallize, and the monomer-dimer equilibrium in solution which resulted in exchange-broadening and disappearance of many peaks from the CTD domain due its reversible CTD-CTD dimerization. Thus, efforts have focused on the structural determinations by crystallography or NMR spectroscopy of isolated domains, viz., the NTD domain (16,17), the dimeric structures of the isolated wild-type CTD domain (8,18), a domain-swapped CTD dimer (19), as well as the structure of the isolated monomeric mutants of the CTD domain (20,21). For the full-length wild-type CA protein, three crystallographic studies of CA dimers have been reported - - a parallel dimer of CA with NTD stabilized by complexation with Fab but with a disordered CTD (22), and antiparallel head-to-tail dimers of CA stabilized by complexation with Fab (23) and with triiodide (24). These dimer structures of the wt CA exhibit intermolecular CA-CA interactions and associated structural perturbations. In this work we report the detailed solution structure of the full-length HIV-1 CA protein in a monomer state. To achieve this goal, we have utilized the strategy of making two critical mutations in the CTD domain of the wild-type (wt) CA that are known to disrupt the infectivity as well as the weak face-to-face dimerization in solution (18), viz., W184A/M185A, resulting in a monomeric form of the CA protein. Most importantly, these two mutations preserve many of the critical properties of the wt CA - - (i) unmyristoylated Gag protein containing these two mutations in CA is assembly-active and forms spherical virus-like particles in vitro and in mammalian cells (25), though the particles tend to be somewhat irregular in size and shape. (ii) Similarly, CA proteins with W184A and/or M185A mutations are assembly active though much less efficiently than the wild type. For example, the M185A-CA mutant has been shown to be assembly active but it assembles about 10 times slower than the wild type (26). The quadruple mutant protein A14C/E45C/W184A/M185A-CA polymerizes under reducing conditions (i.e., in the presence of β-mercaptoethanol to prevent spurious cross-linking of cysteines) and forms long tubes in vitro, though somewhat less efficiently than the wild-type (1) (it is only after polymerization that the cysteines were allowed in this study to oxidize so that stable CA-hexamers with correct cross-linking could be isolated for crystallization). The CTD-CTD dimer interface is preserved between these mutant CA hexamers despite the W184A/M185A mutations (1). Following a suggestion that our double mutant might in fact show such assembly activity at high concentrations (private communication, Peter Prevelige, Jr, 2011), we had undertaken in vitro assembly reactions. The data are shown in Figure 1, demonstrating again, and in agreement with the findings of Pornillos et al., (1) on the quadruple mutant, that our double mutant W184A/M185A-CA is indeed assembly active (but at higher concentrations than the wild type) and its assembly rate is much slower than that of the wild type (estimated to be about 28 times slower based on the data at 60 μM). (iii) The secondary structures of the double mutant Gag and the wt Gag (25) as well as of the double mutant CA and the wild-type CA (20) are also identical, as established by CD on very dilute solutions. (iv) Further, mutations at 184 and 185 which inhibit CTD-CTD dimerization in both CA and in Gag do not significantly alter the affinity of CA to host cell binding partners - - e.g., the binding properties of M185A-CA with prolyl isomerase cyclophilin A (27), and that of Gag polyprotein containing the W184A/M185A– CA domain with human lysyl t-RNA synthetase (28) are similar to that of the respective wild-type molecules. Taken together, the results of the above Gag and capsid assembly reactions, CD data, and binding assays with host cell binding partners demonstrate that the monomeric mutant CA, though not infective, retains many of the critical properties of the wild-type CA even though it exists as a monomer in solution. Residues W184 and M185 in HIV-1 CA appear to be important for infectivity (18), dimerization in solution, for improving the efficiency of assembly, and for providing uniformity in the size and shapes of the assembled capsids. However, as demonstrated by others previously (1) and by us in this work (Figure 1), the capsid protein is assembly active though much less efficiently than the wild type protein when these two residues are replaced by alanine. We note that these two residues are not conserved in other retroviruses (19). Figure 1 In vitro assembly of the HIV-1 capsid protein : (A) wild-type CA (top) and (B) the double mutant W184A/M185A-CA (bottom) at a final concentration of 2.25 M NaCl. The concentrations of the proteins are indicated on the right side of each panel.