HIV-1 Matrix Protein Research Articles

HIV-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly

The HIV-1 matrix (MA) protein is similar to nucleocapsid (NC) proteins in its propensity for self-interaction and association with RNA. Here we report on our finding that replacing MA with NC results in the production of wild type (wt)-level RNA and virus-like particles (VLPs). In contrast, constructs containing MA as a substitute for NC are markedly defective in VLP production and form virions with lower densities than wt, even though their RNA content is over 50% that of wt level. We also noted that a ΔMN mutant lacking both MA and NC produces a relatively higher amount of VLPs than those in which MA was substituted for NC. Although ΔMN contains approximately 30% the RNA of wt, it still exhibits virion densities equal (or very similar) to those of wt. The data suggest that neither NC nor RNA are major virion density determinants. Furthermore, we noted that NC(ZIP)—a NC replacement with a leucine zipper dimerization motif—produces VLPs as efficiently as wt. However, the markedly reduced assembly efficiency of NC(ZIP) is associated with the formation of VLPs with densities slightly lower than those of wt following MA removal, suggesting that (a) MA is required to help the inserted leucine zipper motif perform efficient Gag multimerization, and (b) MA plays a role in the virus assembly process.

HIV-1 Matrix Protein p17 Prevents Loss of CD28 Expression During IL-2–Induced Maturation of Naïve CD8+T Cells

Naïve CD8(+) T cells differentiate into effectors secreting various cytokines that modulate immune functions. A striking finding for most HIV-1-infected patients is that they accumulate CD8(+) T cells belonging to early and intermediate differentiated elements. Structural HIV-1 proteins, and among these the matrix protein p17, have been associated with loss of functional competence by different immune cells. We therefore evaluated the influence of p17 on naïve CD8(+) T-cell activation and maturation. Anti-CD3 mAb preactivation and subsequent IL-2 stimulation are able to drive human naive CD(+) T cells to an effector phenotype characterized, among other features, by downregulation of the co-stimulatory molecule CD28. Strikingly, however, IL-2-induced downmodulation of CD28 was completely prevented by p17, and cells derived from p17-stimulated cultures showed a strong Tc1 polarization that was fourfold higher than that observed in IL-2-stimulated cultures.Moreover, p17 preserved a markedly high proportion of CD8(+) T cells that were able to respond to CD28 triggering with a proinflammatory cytokine storm. Our evidence suggests that p17 has important effects on cytokine polarization and phenotype of terminally differentiated CD8(+) T cells, and that new p17-based therapeutic approaches could control or prevent HIV-1-related immune disorders.

HIV-1 matrix protein p17 induces human plasmacytoid dendritic cells to acquire a migratory immature cell phenotype

Numerical and functional defects in plasmacytoid dendritic cells (pDCs) are an important hallmark of progressive HIV-1 infection, yet its etiology remains obscure. HIV-1 p17 matrix protein (p17) modulates a variety of cellular responses, and its biological activity depends on the expression of p17 receptors (p17Rs) on the surface of target cells. In this study, we show that peripheral blood pDCs express p17Rs on their surface and that freshly isolated pDCs are sensitive to p17 stimulation. Upon p17 treatment, pDCs undergo phenotypic differentiation with up-regulation of CCR7. A chemotaxis assay reveals that p17-treated pDCs migrate in response to CCL19, suggesting that these cells may acquire the ability to migrate to secondary lymphoid organs. In contrast, p17 does not induce release of type I IFN nor does it enhance pDC expression of CD80, CD86, CD83, or MHC class II. Microarray gene expression analysis indicated that p17-stimulated pDCs down-regulate the expression of molecules whose functions are crucial for efficient protein synthesis, protection from apoptosis, and cell proliferation induction. Based on these results, we propose a model where p17 induces immature circulating pDCs to home in lymph nodes devoid of their ability to serve as a link between innate and adaptative immune systems.

The N-Terminal Basic Domain of the HIV-1 Matrix Protein Does Not Contain a Conventional Nuclear Localization Sequence But Is Required for DNA Binding and Protein Self-Association

The HIV p17 or matrix (MA) protein has long been implicated in the process of nuclear import of the HIV genome and thus the ability of the virus to infect nondividing cells such as macrophages. While it has been demonstrated that MA is not absolutely required for this process, debate continues to surround the subcellular targeting properties of MA and its potential contribution to nuclear import of the HIV cDNA. Through the use of in vitro techniques we have determined that, despite the ability of MA to interact with importins, the full-length protein fails to enter the nucleus of cells. While MA does contain a region of basic amino acids within its N-terminus which can confer nuclear accumulation of a fusion protein, we show that this is due to nuclear retention mediated by DNA binding and does not represent facilitated import. Importantly, we show that the 26KK residues of MA, previously thought to be part of a nuclear localization sequence, are absolutely required for a number of MA's functions including its ability to bind DNA and RNA and its propensity to form high-order multimers/protein aggregates. The results presented here indicate that the N-terminal basic domain of MA does not appear likely to play a role in HIV cDNA nuclear import; rather this region appears to be a crucial structural and functional motif whose integrity is required for a number of other roles performed by MA during viral infection.

HIV-1 matrix protein p17 binds to monocytes and selectively stimulates MCP-1 secretion: role of transcriptional factor AP-1

SummaryHIV‐1 matrix protein p17 activates a variety of cell responses which play a critical role in viral replication and infection. Its activity depends on the expression of p17 receptors (p17R) on the surface of target cells. Whether p17 also plays a role in stimulating human monocytes, a major HIV‐1 reservoir, is not known. Here we show that human monocytes constitutively express p17Rs and that p17 selectively triggers these cells to produce MCP‐1. The effect of p17 on MCP‐1 expression was observed at the transcriptional level and was primarily dependent on the activation of the transcription factor AP‐1. p17 increased the binding activity of AP‐1 complexes in a time‐ and dose‐dependent manner. Deletion of the AP‐1 binding sites in the MCP‐1 promoter resulted in the lack of p17‐induced MCP‐1 transcription. In particular, the P3 binding site located between −69 and −63 position seems to be essential to MCP‐1 mRNA induction in p17‐treated monocytes. An ever increasing amount of evidences shows a tight link between biologically dysregulated monocytes, AP‐1 activation, MCP‐1 release and HIV‐1 pathogenesis. Overall our results suggest that p17 may play a critical role in the monocyte‐mediated inflammatory processes, which are suspected to be major precipitating events in AIDS‐defining diseases.

Regulating The Functions of The HIV-1 Matrix Protein

The HIV-1 structural protein matrix (MA) is involved in a number of essential steps during infection and appears to possess multiple, seemingly conflicting targeting signals. Although MA has long been known to be crucial for virion assembly, details regarding this function, and the domains responsible for mediating it, are still emerging. MA has also been implicated in nuclear import of HIV cDNA and is purported to contain a nuclear targeting signal. Little is known about how these opposing plasma membrane and nuclear targeting signals are regulated and which signals predominate at various stages of infection. Additionally, MA has recently been implicated in a number of novel roles during infection including viral entry/uncoating, cytoskeletal-mediated transport, and targeting viral assembly to lipid rafts. Here we discuss our current understanding of MA's functions during infection and explore the recent advancements made in elucidating the mechanism of these processes. It appears that MA possesses a cache of targeting signals that are likely to be regulated throughout the infectious cycle by a combination of structural and biochemical modifications including phosphorylation, myristoylation, and multimerization. The ability of HIV to modify the properties of MA at specific stages of infection is central to the multifunctional behavior of MA and the efficiency of HIV infection. The recently reported success of drugs specifically designed to block MA function (Haffar O, Dubrovsky L, and Lowe R et al. J Virol 2005;79:13028-13036) confirms the importance of this protein for HIV infection and highlights a potentially new avenue in multivalent drug therapy.

Molecular dynamics analysis of HIV-1 matrix protein: Clarifying differences between crystallographic and solution structures

One of the main structural features of the mature HIV-1 virion is the matrix protein (p17). This partially globular protein presents four helixes centrally organized and a fifth one, H5, projecting away from the packed bundle of helixes. Comparison between solution and crystallographic data of p17 indicates a 6 Å displacement of a short 3 10 helix and a partial unfolding of H5 in solution related to crystal. While the behavior of the 3 10 helix has been previously addressed to virion assembly, the relevance and origin of H5 partial unfolding is possibly related to the contacts between p17 and other viral elements, such as p24. In this context, we present a 40 ns conformational sampling of monomeric p17 using molecular dynamics simulations. The performed simulations presented a progressive conversion of the p17 crystallographic structure to the NMR conformation, suggesting that the biological form of this protein may have its C-terminal portion partially unfolded.

HIV-1 Matrix Protein p17 Modulatesin VivoPreactivated Murine T-Cell Response and Enhances the Induction of Systemic and Mucosal Immunity Against Intranasally Co-administered Antigens

HIV-1 p17 is a viral cytokine that acts on preactivated, but not on resting, human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication, after binding to a cellular receptor (p17R). Here, we demonstrate that p17Rs are expressed on activated murine T cells, which respond to p17 stimulation similarly to their human counterpart. We developed a mouse model of abortive HSV-1 infection to induce T cell activation in vivo. Preactivated cells expressed p17Rs and were highly susceptible to p17 stimulation, which triggered proinflammatory cytokines release and promoted CD4+ T cell survival and expansion. Coculture of in vivo activated splenocytes with macrophages in the presence of p17 further increased their ability to produce IFN-gamma. The presence of macrophages and activated T cells at mucosal sites prompted us to investigate the immunomodulatory activities of p17 in vivo. Intranasal coadministration of p17 with beta-galactosidase (beta-gal) resulted in improved beta-gal specific cellular and humoral immune responses at systemic and mucosal levels. It is well established that HIV-1 replication is driven in an autocrine/paracrine manner by endogenously produced proinflammatory cytokines. Our results highlight the role of p17 in sustaining cellular activation and inflammation, thereby promoting a permissive microenvironment for HIV-1 replication. In addition, p17 is a promising candidate antigen, exhibiting immunomodulatory/adjuvant properties, that need to be exploited in the development of HIV/AIDS vaccines.

In Vivo Administration of Replication-Deficient Mutant HSV-1 Targets Professional APCs and Induces Efficient CD4+ T Helper Responses

Both neutralizing antibodies and cytotoxic T cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. These findings have critical implications in the design of vaccination strategies aimed at triggering and sustaining antigen specific CD4+ in addition to CD8+ effector immune responses. Here we show that a recombinant replication-deficient HSV-1 vector encoding the HIV-1 matrix protein p17 (T0-p17) is capable to infect professional APCs in vitro and in vivo without interfering with the endogenous MHC class II processing of the transgene encoded antigen. Moreover, we show that injection of T0-p17 in the mouse dermis generates a strong p17specific CD4+ T helper response preceding both cytotoxic and humoral responses. Importantly, T0-p17 infected peritoneal macrophages were capable to trigger a longlasting expansion of p17-specific CD4+ T cells in vitro. Because of their capability to infect professional APCs without interfering with their biological functions, replication-deficient HSV vectors are appealing candidates for the development of vaccines able to trigger strong T helper responses. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The HIV-1 matrix protein p17 can be efficiently delivered by intranasal route in mice using the TLR 2/6 agonist MALP-2 as mucosal adjuvant

The HIV-1 matrix protein p17 is a structural protein essential in the life cycle of HIV, by acting as a virokine/immunomodulator that supports viral replication and spreading. The presence of p17-specific antibodies and CTL responses correlates with slower progression to AIDS. Intranasal vaccination with p17 and the TLR2/6 agonist MALP-2 stimulates strong humoral and cellular immune responses at systemic and mucosal levels. The antibodies blocked p17 binding to its receptor, which is a critical step for the exertion of its virokine activity. Our results suggest that p17 and MALP-2 are attractive candidates for incorporation in mucosal vaccines against HIV/AIDS.

Human immunodeficiency virus type 1 KK 26–27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import

We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26–27 in MA reported nuclear localization signal (NLS)-1 [ Haffar, O.K., Popov, S., Dubrovsky, L., Agostini, I., Tang, H., Pushkarsky, T., Nadler, S.G., Bukrinsky, M., 2000. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J. Mol. Biol. 299 (2): 359–368] impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.

Polymorphism of HIV-1 gag (p17) gene from female sex workers in Calcutta, India

HIV-1 subtype C is the major subtype in India as evidenced from the analysis of specific regions within envelope and gag gene. The matrix protein (p17) of HIV-1 which is involved in several functions like the viral RNA transport, nuclear localization, assembly of pre-integration complex into host nucleus has been used to study the strain diversity among female sexworkers in Calcutta. The gene encoding for the HIV-1 matrix protein, p17 was amplified by nested polymerase chain reaction (PCR) from blood samples of HIV-1 seropositive female sex workers (FSW) in Calcutta, India. Genes of twenty-two samples were sequenced and the phylogenetic analysis with different global strains showed that the majority (seventeen) was clustered with Indian type C. A few samples were found to be close to other C subtypes isolated from South Africa, China and Myanmar. The comparison of Calcutta samples with the samples from other regions of India along with other non-asian subtype C sequences clearly revealed a different cluster of Indian sequences. The two samples, cal 242 and cal 709 was found to be the most divergent type and showed close relatedness with African C subtypes.

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: structural and mechanistic implications of p17 myristoylation.

The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this "myristoyl switch" hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.

Entropic switch regulates myristate exposure in the HIV-1 matrix protein.

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (K(assoc) = 2.5 +/- 0.6 x 10(8) M(-2)). The equilibrium constant is shifted approximately 20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.

Simple mimetics of a nuclear localization signal (NLS).

[reaction: see text] Molecular modeling was used to design mimetics of the HIV-1 matrix protein nuclear localization signal (NLS) in which a scaffold of two resorcinol units joined by a diamide linker presents 3-aminopropyl ethers in place of lysine side chains. Prospective mimetics with linkers of 6, 8, 10, or 12 atoms were synthesized and compared in a competition assay for binding to the nuclear import receptor subunit karyopherin alpha, showing the 10-atom linker to be best and shorter ones ineffective.

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor.

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor alpha and IFN-gamma released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-gamma and tumor necrosis factor alpha, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH(2)-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein.

Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.

Immunologic cross-reaction between HIV type 1 p17 and Mycoplasma hyorhinis variable lipoprotein.

Monoclonal antibodies directed against the HIV-1 matrix protein p17 that react with a component present on the surface of HIV-1-infected cells have previously been described. In this study we show that one of these monoclonal antibodies binds to persistently HIV-1-infected cell lines that are coinfected with Mycoplasma hyorhinis, but not to cell lines that are uninfected with mycoplasma. Mycoplasma-infected cells secrete HIV-1 at a higher rate, have a slight increase in cell surface expression of gp120 and gp41, and are less sensitive to immunotoxins than uninfected cells. The anti-p17 antibody binds to a protein of M. hyorhinis grown in cell-free culture. The variable expression and size of the protein among strains is typical of the variable lipoprotein (Vlp) system of M. hyorhinis. Confirmation of the reactivity of the antibody with a Vlp was provided by demonstrating its specific binding to recombinant VlpF expressed in E. coli, and to a synthetic peptide representing the carboxy-terminal region of VlpF, but not to other recombinant Vlp products or peptides. This is a true cross-reaction because the antibody also binds to recombinant p17 expressed in E. coli and the binding is inhibited by the VlpF peptide. These analyses highlight the potential of mycoplasma contamination of tissue culture cell lines to cause anomalous results. With regard to HIV-1, mycoplasma infection of cells results in increased rates of virus secretion, and introduces a potential confounding immunologic cross-reaction as well. The existence of a cell surface form of p17 is unlikely.

Organization of HIV-1 Capsid Proteins on a Lipid Monolayer

In an in vitro system that mimics the assembly of immature human immunodeficiency virus (HIV) particles, ordered arrays of HIV-1 capsid (CA) proteins encoded by the viral gag gene have been obtained by incubation of histidine-tagged capsid proteins (His-HIVCA) beneath lipid monolayers containing the nickel-chelating lipid, 1,2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'-N-(5-amino-1- carboxypentyl)iminodiacetic acid)propyl ether. The membrane-bound His-HIVCA proteins formed small crystalline arrays of primitive (p1) unit cells with dimensions of a = 74.2 A, b = 126.2 A, gamma = 89.3 degrees. The image-analyzed two-dimensional projection of His-HIVCA assemblies shows a cage-like lattice, consisting of hexamer and trimer units, surrounding protein-free cage holes. The hexamer-coordinated cage holes of 26.3-A diameter are spaced at 74. 2-A intervals: these distances, and the hexamer-trimer arrangement, are consistent with previous, lower resolution studies on immature HIV-1 virus particles produced in vivo. Additionally, HIV-1 matrix protein trimer unit structures align to the His-HIVCA trimer units such that residues previously shown to interact with the HIV-1 gp120/gp41 envelope protein complex are oriented toward the hexamer cage holes. Our results form a bridge between results from conventional methods for the analysis of HIV particle structure.