Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

Inhibition of wild-type and mutant human immunodeficiency virus type 1 proteases by GW0385 and other arylsulfonamides

The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM.     

DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.     

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 tat protein

The human immunodeficiency virus type 1 Tat protein is a potent activator of viral gene expression and replication. Tat can also affect the expression of cellular genes including cytokines, extracellular matrix proteins, enzymes degrading the basement membrane and cell cycle-related proteins, and can regulate cellular functions such as growth, migration and angiogenesis. In addition, under certain circumstances, Tat may have tumorigenic effects. These activities of Tat appear to be mediated by different mechanisms such as the transactivation of cellular gene expression or the interaction of extracellular Tat with the cell membrane through both receptor-mediated and nonreceptor-mediated interactions. Deregulation of cellular gene expression and function by Tat cause abnormalities which may participate in AIDS pathogenesis and in the development of AIDS-associated disorders.     

DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets

Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.     

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells.

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.     

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.     

Blockade of human immunodeficiency virus type 1 expression by caveolin-1

Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.     

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.     

Phosphorylation of the rev gene product of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.