Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41

Two α-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01_AE, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants.     

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20-70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.     

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: effects on virion morphogenesis and RNA maturation

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag–Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC₉₀), most of the Gag and Gag–Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.     

The T12I mutation within the SP1 region of Gag restricts packaging of spliced viral RNA into human immunodeficiency virus type 1 with mutated RNA packaging signals and mutated nucleocapsid sequence

Specific packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA packaging signals located within the 5' un-translated region (5' UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5' UTR sequence, such as DeltaGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Delta(306-325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA packaging that is neither pertained to the specific viral RNA packaging signal nor to the NC sequence.     

Downregulation of human immunodeficiency virus type 1 Gag expression by a gp41 cytoplasmic domain fusion protein

The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, beta-galactosidase (beta-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli beta-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.     

Depletion of cellular cholesterol inhibits membrane binding and higher-order multimerization of human immunodeficiency virus type 1 Gag

Recent studies have suggested that the plasma membrane contains cholesterol-enriched microdomains known as lipid rafts. HIV-1 Gag binds raft-rich regions of the plasma membrane, and cholesterol depletion impairs HIV-1 particle production. In this study, we sought to define the block imposed by cholesterol depletion. We observed that membrane binding and higher-order multimerization of Gag were markedly reduced upon cholesterol depletion. Fusing to Gag a highly efficient, heterologous membrane-binding sequence reversed the defects in Gag-membrane binding and multimerization caused by cholesterol depletion, indicating that the impact of reducing the membrane cholesterol content on Gag-membrane binding and multimerization can be circumvented by increasing the affinity of Gag for membrane. Virus release efficiency of this Gag derivative was minimally affected by cholesterol depletion. Altogether, these results are consistent with the hypothesis that cholesterol-enriched membrane microdomains promote HIV-1 particle production by facilitating both Gag-membrane binding and Gag multimerization.     

Envelope glycoproteins of human immunodeficiency virus type 1 variants issued from mother-infant pairs display a wide spectrum of biological properties

Several studies have shown that the early virus population present in HIV-1 infected infants usually is homogeneous when compared to the highly diversified viral population present at delivery in their mothers. We explored the antigenic and functional properties of pseudotyped viruses expressing gp120 encoded by env clones issued from four mother-infant pairs infected by CRF01_AE viruses. We compared their sensitivity to neutralization and to entry inhibitors, their infectivity levels and the Env processing and incorporation levels. We found that both transmitted viruses present in infants and the variants present in their chronically infected mothers display a wide spectrum of biological properties that could not distinguish between them. In contrast, we found that all the transmitted viruses in the infants were more sensitive to neutralization by PG9 and PG16 than the maternal variants, an observation that may have implications for the development of prophylactic strategies to prevent mother-to-child transmission.     

Serum antibody pattern, antigenemia, and virus isolation in infants born to mothers seropositive for human immunodeficiency virus type 1

Forty children born to mothers seropositive for the human immunodeficiency virus type 1 (HIV-1) followed-up to 15 months after birth were studied by means of serum antibody patterns to individual viral polypeptides, by the presence of detectable levels of viral core antigen (p24) and virus in serum and peripheral blood lymphocytes, and by total lymphocyte counts and T4/T8 lymphocyte ratios. The results obtained indicate that a persistent antigenemia is significantly associated with positive virus isolation from peripheral blood lymphocytes and with changes in the intensity of antibody reaction to core (p24, p17) and pol (p31) antigens. Six children (15%) presented unequivocal signs of HIV-1 infection and five also had signs of immune system involvement.     

Drug-associated changes in amino acid residues in Gag p2, p7(NC), and p6(Gag)/p6(Pol) in human immunodeficiency virus type 1 (HIV-1) display a dominant effect on replicative fitness and drug response

Regions of HIV-1 gag between p2 and p6^Gag/p6^Pol, in addition to protease (PR), develop genetic diversity in HIV-1 infected individuals who fail to suppress virus replication by combination protease inhibitor (PI) therapy. To elucidate functional consequences for viral replication and PI susceptibility by changes in Gag that evolve in vivo during PI therapy, a panel of recombinant viruses was constructed. Residues in Gag p2/p7^NC cleavage site and p7^NC, combined with residues in the flap of PR, defined novel fitness determinants that restored replicative capacity to the posttherapy virus. Multiple determinants in Gag have a dominant effect on PR phenotype and increase susceptibility to inhibitors of drug-resistant or drug-sensitive PR genes. Gag determinants of drug sensitivity and replication alter the fitness landscape of the virus, and viral replicative capacity can be independent of drug sensitivity. The functional linkage between Gag and PR provides targets for novel therapeutics to inhibit drug-resistant viruses.     

The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization

The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~ 100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9). However, changes at position 675 in conjunction with Thr to Ala at position 569 increased the neutralization sensitivity to all gp41 and gp120 MAbs and plasma, in some cases by more than 1000-fold. Interestingly, the T569A change had a dramatic effect on b12 binding, but no effect on neutralization sensitivity. This finding suggests that antibody neutralization may occur through a multi-step pathway that includes distinct changes in envelope conformation that may affect binding but not neutralization susceptibility.     

Contribution of the Gag-Pol transframe domain p6* and its coding sequence to morphogenesis and replication of human immunodeficiency virus type 1

The human immunodeficiency virus type-1 (HIV-1) transframe domain p6* is located between the nucleocapsid protein (NC) and the protease (PR) within the Gag-Pol precursor. This flexible, 68-amino-acid HIV-1 p6* domain has been suggested to negatively interfere with HIV PR activity in vitro proposing a contribution of either the C-terminal p6* tetrapeptide, internal cryptic PR cleavage sites, or a zymogen-related mechanism to a regulated PR activation. To assess these hypotheses in the viral context, a series of recombinant HX10-based provirus constructs has been established with clustered amino acid substitutions throughout the entire p6* coding sequence. Comparative analysis of the mutant proviral clones in different cell culture systems revealed that mutations within the well-conserved amino-terminal p6* region modified the Gag/Gag-Pol ratio and thus resulted in the release of viruses with impaired infectivity. Clustered amino acid substitutions destroying (i) the predicted cryptic PR cleavage sites or (ii) homologies to the pepsinogen propeptide did not influence viral replication in cell culture, whereas substitutions of the carboxyl-terminal p6* residues 62 to 68 altering proper release of the mature PR from the Gag-Pol precursor drastically reduced viral infectivity. Thus, the critical contribution of p6* and overlapping cis-acting sequence elements to timely regulated virus maturation and infectivity is closely linked to precise ribosomal frameshifting and proper N-terminal release of the viral PR from the Gag-Pol precursor, clearly disproving the hypothesis that sequence motifs in the central part of p6* modulate PR activation and viral infectivity.     

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3-azido-3-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.     

Lack of predictive for progression of dissociated circulating P24 antigen in human immunodeficiency virus type 1-infected black patients

In human immunodeficiency virus type 1 (HIV)-1-infected Black people, the circulating p24 antigen is hidden frequently in immune complexes, because of high titers of serum anti-p24 antibodies. In order to evaluate the prognostic values for progression of free and dissociated serum p24 antigen in Black people, sera from 45 HIV-1-infected Black patients, all at non-AIDS stages, were evaluated prospectively for p24 antigen by several assays: circulating free p24 antigen was measured by immunocapture ELISA only (method 1) and with ELAST^TM amplification (method 2), and dissociated p24 antigen determined after glycine-HCl pretreatment of serum, by immunocapture ELISA only (method 3) and with ELAST^TM amplification (method 4). Serum CD4 and CD8 cell counts, β₂-microglobulin, and total IgA were determined also at least twice a year. Clinical events for AIDS were those included in the 1986 CDC classification for HIV infection. At entry, p24 antigen was found in 3 (6.7%) patients by method 1, in 7 (15.6%) by method 2, in 14 (31.1%) by method 3, and in 22 (48.9%) by method 4. Methods 3 and 4 were more sensitive than method 1 (P < 0.001) and method 2 (P < 0.001). The mean follow-up was 30 months. The free symptom survival times (mean ± SD months) were significantly lower in patients being p24 antigen positive by method 1 [(+) 33 ± 27 vs. (−) 61 ± 15, P = 0.03], but they were similar in patients positive and in those negative for p24 antigen determined by method 2 [(+) 71 ± 17 vs. (−) 74 ± 9, P = 0.54], method 3 [(+) 76 ± 12 vs. (−) 69 ± 13.2, P = 0.80], and method 4 [(+) 79 ± 9 vs. (−) 63 ± 7, P = 0.71]. At 24 months, p24 antigen positivity did not correlate either with CD4 or CD4/CD8 slops, nor with β₂-microglobulin or IgA variations. By contrast, a CD4 cell count below 200/mm³ at entry was significantly associated with disease progression. In conclusion, dissociated p24 antigenemia does not appear as a useful surrogate marker for progression in HIV-1-infected Black people. © 1996 Wiley-Liss, Inc.     

Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D)

We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry. Neither escape mutant could replicate detectably in peripheral blood mononuclear cells (PBMC) from two donors homozygous for the CCR5-Delta32 allele and both were insensitive to the CXCR4-specific inhibitor, AMD3100. Hence, the SCH-D escape mutants retained the R5 phenotype. One of the resistant isolates was, however, capable of replication in U87.CD4.CXCR4 cells and, after expansion in those cells, was sensitive to AMD3100 in primary CD4+ T-cells. Hence, some X4 variants may be present in this escape mutant swarm. A notable observation was that the SCH-D escape mutants were also cross-resistant to PSC-RANTES and AOP-RANTES, chemokine derivatives that are reported to down-regulate cell surface CCR5 almost completely. However, the extent to which CCR5 is down-regulated was dependent upon the detection MAb. Hence, the escape mutants may be using a CCR5 configuration that is only detected by some anti-CCR5 MAbs. Finally, two SCH-D-resistant clonal viruses revealed no amino acid changes in the gp120 V3 region relative to the parental viruses, in marked contrast to clones resistant to the AD101 small molecule CCR5 inhibitor that possess 4 such sequence changes. Several sequence changes elsewhere in gp120 (V2, C3 and V4) were present in the SCH-D-resistant clones. Their influence on the resistant phenotype remains to be determined.