Sites, mechanism of action and lack of reversibility of primate lentivirus inactivation by preferential covalent modification of virion internal proteins.

By exploiting the intrinsic chemistry of retroviruses, we have developed a novel method for generating whole inactivated virion vaccine immunogens with functional envelope glycoproteins. The method takes advantage of the fact that the internal proteins of retroviruses are adapted to the intracellular (reducing) environment, and have cysteine residues present in thiol-form (S-H), while the surface proteins of retroviruses (the envelope glycoproteins SU and TM) are adapted to the (oxidizing) environment of the extracellular milieu, and have their cysteines present as disulfides (S-S). Treatment of retroviral virions with appropriate mild oxidizing agents thus results in preferential covalent modification and functional inactivation of key S-H-containing internal viral proteins, such as the nucleocapsid (NC) protein, that are required for infectivity, while the envelope glycoproteins with their disulfide bonded cysteines remain unaffected. This treatment thus results in virions that do not retain detectable infectivity, but preserves the conformational and functional integrity of the envelope glycoproteins. We have extensively used the disulfide reagent 2,2'-dithiodipyridine (aldrithiol-2, AT-2) to inactivate HIV and SIV via this mechanism and such inactivated virions appear to be a promising vaccine immunogen based on macaque studies. We have biochemically characterized the targets and mechanisms of inactivation involved in AT-2 treatment of virions, and investigated the kinetics of inactivation. Although extremely unlikely under physiological conditions, reversibility of this type of inactivation is a theoretical concern. We have therefore conducted a series of in vitro experiments, in cell free systems and in cell culture, to evaluate this possibility. The results indicate that as judged by both biochemical and biological (infectivity) criteria, inactivation by AT-2 does not appear to be reversible under conditions likely to obtain in vivo.

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.

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4.

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.

Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus.

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55 degrees C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37 degrees C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.