Molecular Determinants that Regulate Plasma Membrane Association of HIV-1 Gag

Human immunodeficiency virus type 1 assembly is a multistep process that occurs at the plasma membrane (PM). Targeting and binding of Gag to the PM are the first steps in this assembly process and are mediated by the matrix domain of Gag. This review highlights our current knowledge on viral and cellular determinants that affect specific interactions between Gag and the PM. We will discuss potential mechanisms by which the matrix domain might integrate three regulatory components, myristate, phosphatidylinositol-(4,5)-bisphosphate, and RNA, to ensure that human immunodeficiency virus type 1 assembly occurs at the PM.     

Gag-Pol Processing during HIV-1 Virion Maturation: A Systems Biology Approach

Proteolytic processing of Gag and Gag-Pol polyproteins by the viral protease (PR) is crucial for the production of infectious HIV-1, and inhibitors of the viral PR are an integral part of current antiretroviral therapy. The process has several layers of complexity (multiple cleavage sites and substrates; multiple enzyme forms; PR auto-processing), which calls for a systems level approach to identify key vulnerabilities and optimal treatment strategies. Here we present the first full reaction kinetics model of proteolytic processing by HIV-1 PR, taking into account all canonical cleavage sites within Gag and Gag-Pol, intermediate products and enzyme forms, enzyme dimerization, the initial auto-cleavage of full-length Gag-Pol as well as self-cleavage of PR. The model allows us to identify the rate limiting step of virion maturation and the parameters with the strongest effect on maturation kinetics. Using the modelling framework, we predict interactions and compensatory potential between individual cleavage rates and drugs, characterize the time course of the process, explain the steep dose response curves associated with PR inhibitors and gain new insights into drug action. While the results of the model are subject to limitations arising from the simplifying assumptions used and from the uncertainties in the parameter estimates, the developed framework provides an extendable open-access platform to incorporate new data and hypotheses in the future.     

Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P₂ and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P₂-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNA^Pro, but not that of tRNA^Lys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNA^Lys3 showed greater inhibition of MA membrane binding than full-length tRNA^Pro. While transplantation of the D loop sequence of tRNA^Lys3 into tRNA^Pro resulted in a modest increase in the inhibitory effect relative to WT tRNA^Pro, replacing the entire D arm sequence with that of tRNA^Lys3 was necessary to confer the full inhibitory effects upon tRNA^Pro. Together, these results demonstrate that the D arm of tRNA^Lys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.     

Huwe1, a novel cellular interactor of Gag-Pol through integrase binding, negatively influences HIV-1 infectivity

Integration, an indispensable step for retrovirus replication, is executed by integrase (IN), which is expressed as a part of a Gag-Pol precursor. Although mechanistic detail of the IN-catalyzed integration reaction is well defined, numerous evidence have demonstrated that IN is involved in multiple steps of retrovirus replication other than integration. In this study, Huwe1, a HECT-type E3 ubiquitin ligase, was identified as a new cellular interactor of human immunodeficiency virus type 1 (HIV-1) IN. The interaction was mediated through the catalytic core domain of IN and a wide-range region of Huwe1. Interestingly, although depletion of Huwe1 in target cells did not affect the early phase of HIV-1 infection in a human T cell line, we found that infectivity of HIV-1 released from the Huwe1 knockdown cells was significantly augmented more than that of virus produced from control cells. The increase in infectivity occurred in proviral DNA synthesis. Further analysis revealed that Huwe1 interacted with HIV-1 Gag-Pol precursor protein through an IN domain. Our results suggest that Huwe1 in HIV-1 producer cells has a negative impact on early post-entry events during the next round of virus infection via association with an IN region of Gag-Pol.     

Human Endogenous Retrovirus K Gag Coassembles with HIV-1 Gag and Reduces the Release Efficiency and Infectivity of HIV-1

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.     

Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.     

Conformation of the HIV-1 Gag protein in solution

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.     

Ty1 Gag Enhances the Stability and Nuclear Export of Ty1 mRNA

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus‐like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1‐less strain expressing galactose‐inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense‐mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.