The work in my laboratory is focused on understanding a variety of aspects of HIV-1 assembly, release, and maturation.  The major areas of investigation are described briefly below.

1.  The subcellular targeting of HIV-1 assembly.  The production of retrovirus particles from infected cells is mediated by the Gag precursor protein.  Following its synthesis in the cytosol, Gag is rapidly and specifically transported to the site of virus assembly.  The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood.  Based on the analysis of mutant HIV-1 Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane.  We recently determined [Ono & Freed, J. Virol., 2004 (PDF - 4759KB)] that, in HeLa and T cells, the MA-mutant Gags that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB).  Interestingly, we find that, in primary human macrophages, both wild type (WT) and MA-mutant Gag proteins are targeted specifically to the MVB.  These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages, and that particle release in this cell type may follow an exosomal pathway.  To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6^Gag and MVB sorting machinery (e.g., Tsg101), we examined the targeting and assembly of Gag mutants lacking p6.  Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and Tsg101 is not required for Gag targeting to the MVB.  These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.  Recent studies in the lab have provided evidence of a role for specific phosphoinositides in HIV-1 Gag targeting [Ono et al., PNAS, 2004 (PDF - 886KB)].

2.  Role of plasma membrane rafts in HIV-1 replication.  We and others have observed that retroviral Gag proteins, rather than being uniformly distributed at the plasma membrane, concentrate in discrete regions at the cell surface.  Since these presumed centers of assembly are likely to be enriched for host proteins that play an active role in virus assembly, Env incorporation, and particle release, it will be very important to determine the composition of these regions.  Much excitement and controversy have been generated by the realization that the plasma membrane, rather than being a uniform sea of lipid, contains a variety of microdomains with specific lipid and protein compositions.  Of particular interest has been the cholesterol/glycosphingolipid-enriched "rafts."  We have demonstrated [Ono & Freed, PNAS, 2001 (PDF - 353KB)] that HIV-1 Gag associates with rafts, and that disruption of these lipid domains with cholesterol-depleting agents markedly and specifically suppresses virus particle release.  We also observe that virions produced from cholesterol-depleted cells display impaired infectivity.  A top priority will be to develop a full understanding of the role rafts play throughout the virus replication cycle.

3.  Role of the HIV-1 matrix protein and the gp41 Env glycoprotein in Env/Gag interactions and Env incorporation.  A critical step in HIV-1 assembly involves the incorporation of the Env glycoproteins into budding particles.  For a number of years, we have been investigating the mechanism by which this incorporation takes place.  We demonstrated the importance of the long cytoplasmic tail of the transmembrane glycoprotein in Env incorporation, and described the cell-type-dependent nature of this function [Murakami & Freed, PNAS, 2000 (PDF - 225KB); Murakami & Freed, J. Virol., 2000 (PDF - 530KB)].  We also demonstrated that uncleaved Gag suppresses Env-mediated fusion activity, indicating a link between virus maturation and Env function [Murakami et al., J. Virol., 2004 (PDF - 266KB)].  Ongoing research is aimed at biochemically characterizing the interaction between Gag and Env, and identifying host protein(s) involved in the Env incorporation process.

4.  Viral and host factors in HIV-1 budding.  Early work from my lab indicated that deletion of the p6 domain of Gag markedly inhibits virus particle production from Gag-expressing HeLa cells, and that this virus release activity maps to a short motif in p6 with the sequence Pro-Thr-Ala-Pro (PTAP) [Huang et al., J. Virol., 1995 (PDF - 913KB)].  Electron microscopy indicated that p6 mutation blocks a very late step in virus release, such that p6-mutant particles fail to bud and remain tethered to the plasma membrane.  Domains with analogous virus release functions, now collectively referred to as "late" or "L" domains, have been identified in the Gag proteins of a number of other retroviruses [Freed, J. Virol., 2002 (PDF - 726KB)].  Three different sequence classes of L domains have been defined: PTAP, Pro-Pro-Pro-Tyr (PPPY), and Tyr-Pro-Asp-Leu (YPDL).

The cellular protein Tsg101, which plays a crucial role in the endosomal sorting pathway, binds HIV-1 Gag in a p6-dependent fashion.  We examined the impact of overexpressing the Gag-binding region of Tsg101 on HIV-1 particle assembly and release.  Intriguingly, we observed that this domain (referred to as TSG-5') potently and specifically inhibits virus production by blocking budding [Demirov et al., PNAS 2002 (PDF - 400KB)].  These results indicate that Tsg101 plays a central role in HIV-1 budding, and also demonstrate that Tsg101 derivatives can act as potent and specific inhibitors of HIV-1 replication.  To elucidate the role of Tsg101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release.  A mutation in TSG-5' that disrupts the Gag/Tsg101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release.  We also determined the effect of overexpressing a panel of truncated Tsg101 derivatives and full-length Tsg101 (TSG-F) on virus budding [Goila-Gaur et al., J. Virol., 2003 (PDF - 3180KB)].  Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding.  Furthermore, overexpression of the C-terminal portion of Tsg101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus.  Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype.  We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery.  We have extended these studies to examine the effect of a number of budding inhibitors on the release of equine infectious anemia virus particles bearing each of the three known classes of L domain (i.e., PTAP, PPPY, and YPDL) [Shehu-Xhilaga et al., J. Virol., 2004 (PDF - 928KB)].  The results highlight the importance of the cellular endosomal sorting machinery in retrovirus budding and indicate that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.

5.  Mechanism of action of PA-457, a novel inhibitor of HIV-1 maturation.  During or shortly after release from the cell, the viral protease cleaves the Gag and GagPol polyprotein precursors to their mature Gag and Pol products.  Gag and GagPol cleavage by protease results in a morphological transition of the particle from immature (displaying a doughnut-shaped morphology) to mature (bearing a condensed, conical core).  In collaboration with Panacos Pharmaceuticals, we have been characterizing the mechanism by which the betulinic acid derivative PA-457 disrupts HIV-1 maturation.  We show that PA-457 potently inhibits replication of HIV-1 by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24).  We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane.  Consistent with the effect on Gag processing, we demonstrate that passaging of WT HIV-1 in the presence of the compound generates a PA-457-resistant virus encoding a single amino acid substitution at the p25 to p24 cleavage site [Li et al., PNAS, 2003 (PDF - 383KB)].  We are currently defining further the target and mechanism of action of PA-457 through mutagenesis and isolation and characterization of a large panel of PA-457-resistant isolates.

Last modified: 5 August 2008