Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

UNLABELLED: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE: Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.

Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.

HIV-1 inactivation by 4-vinylpyridine is enhanced by dissociating Zn(2+) from nucleocapsid protein.

Selective inactivation of critical cysteine residues in human immunodeficiency virus type one (HIV-1) was observed after treatment with 4-vinylpyridine (4-VP), with and without the membrane-permeable metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Chromatographic analysis showed that cysteines contained within nucleocapsid zinc fingers, in the context of whole virus or purified protein, were essentially unreactive, but became reactive when a chelator was included. Virus treated with 4-VP showed only a modest decrease in infectivity; after TPEN addition, nearly complete inactivation of HIV-1 occurred. Similarly, quantitation of viral DNA products from 4-VP-treated virus infections showed no significant effects on reverse transcription, but did show a 14-fold reduction in proviruses; when TPEN was added, a 10(5)-fold decrease in late reverse transcription products was observed and no proviruses were detected. Since 4-VP effectiveness was greatly enhanced by TPEN, this strongly suggests that modification of nucleocapsid zinc fingers is necessary and sufficient for HIV-1 inactivation by sulfhydryl reagents.

Mutational analysis of the C-terminal gag cleavage sites in human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6(Gag) proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9(NC/SP2) protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8(SP2/p6) or p15(NC/SP2/p6) protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6(Gag) site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6(Gag) and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9(NC/SP2) species in some aspect of integration.

HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.

HIV-1 virions are highly enriched in cholesterol relative to the cellular plasma membrane. We recently reported that a cholesterol-binding compound, amphotericin B methyl ester (AME), blocks HIV-1 entry and that single amino acid substitutions in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 confer resistance to AME. In this study, we defined the mechanism of resistance to AME. We observed that the gp41 in AME-resistant virions is substantially smaller than wild-type gp41. Remarkably, we found that this shift in gp41 size is due to cleavage of the gp41 cytoplasmic tail by the viral protease. We mapped the protease-mediated cleavage to two sites in the cytoplasmic tail and showed that gp41 truncations in this region also confer AME resistance. Thus, to escape the inhibitory effects of AME, HIV-1 evolved a mechanism of protease-mediated envelope glycoprotein cleavage used by several other retroviruses to activate envelope glycoprotein fusogenicity. In contrast to the mechanism of AME resistance observed for HIV-1, we demonstrate that simian immunodeficiency virus can escape from AME via the introduction of premature termination codons in the gp41 cytoplasmic tail coding region. These findings demonstrate that in human T cell lines, HIV-1 and simian immunodeficiency virus can evolve distinct strategies for evading AME, reflecting their differential requirements for the gp41 cytoplasmic tail in virus replication. These data reveal that HIV-1 can escape from an inhibitor of viral entry by acquiring mutations that cause the cytoplasmic tail of gp41 to be cleaved by the viral protease.

Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture.

Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli. To facilitate a large-scale production of recombinant His-GRFT, we tested different expression conditions to optimize the expression in the cytoplasm of E. coli to increase the overall production of soluble His-GRFT. Attempts to express His-GRFT in shake flask cultures resulted in a modest yield of soluble His-GRFT, with a large accumulation of the protein in inclusion bodies. The use of a fermenter and of a rich, auto-inducing medium allowed the total amount of His-GRFT per liter to be increased by about 45-fold, with approximately 70% of the protein expressed in the soluble fraction. N-terminal sequencing and MALDI-TOF analyses of the recombinant His-GRFT confirmed that the initial methionine residue was cleaved off. Recombinant His-GRFT showed equivalent activity with natural GRFT, both in respect to gp120-binding characteristics as well as anti-HIV activity. Size-exclusion chromatography analysis showed that both native GRFT and recombinant His-GRFT existed as homodimers in solution. The expression system described in this work provides a basis for the mass production of GRFT to allow further studies of the protein and investigation of therapeutic and preventive strategies against HIV.

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp.

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.

Retroviruses have differing requirements for proteasome function in the budding process.

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.

Equine infectious anemia virus and the ubiquitin-proteasome system.

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant.

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium.

A new anti-HIV protein, scytovirin, was isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein displayed potent anticytopathic activity against laboratory strains and primary isolates of HIV-1 with EC50 values ranging from 0.3 to 22 nM. Scytovirin binds to viral coat proteins gp120, gp160, and gp41 but not to cellular receptor CD4 or other tested proteins. This unique protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds.