Effect of beta-chemokines on human immunodeficiency virus type 1 replication, binding, uncoating, and CCR5 receptor expression in human monocyte-derived macrophages.

OBJECTIVES: We examined the effect and time of addition of beta-chemokines on human immunodeficiency virus type 1 (HIV-1) replication, binding, and uncoating in human macrophages and measured CCR5 receptor expression during virus binding and uncoating. METHODS: Macrophages were treated with beta-chemokines before infection, at infection, or postinfection, and virus replication was determined by p24 antigen level. Binding and uncoating of 35[S]-methionine-labeled HIV-1 was measured. CCR5 expression was determined by flow cytometry. RESULTS: The beta-chemokines potently inhibited virus replication. The strongest inhibition occurred when cultures were pretreated and maintained with beta-chemokines. Beta-chemokines also caused strong inhibition of viral uncoating and a considerable decrease in CCR5 expression during uncoating. CONCLUSIONS: CCR5 receptors appear to be internalized and recycled to the cell surfaces during HIV entry. The down-regulation of CCR5 expression by beta-chemokines during virus uncoating probably accounts for the reduction in virus uncoating (entry) and hence in virus replication.     

Clinical significance of human immunodeficiency virus type 1 replication fitness

The relative fitness of a variant, according to population genetics theory, is that variant's relative contribution to successive generations. Most drug-resistant human immunodeficiency virus type 1 (HIV-1) variants have reduced replication fitness, but at least some of these deficits can be compensated for by the accumulation of second-site mutations. HIV-1 replication fitness also appears to influence the likelihood of a drug-resistant mutant emerging during treatment failure and is postulated to influence clinical outcomes. A variety of assays are available to measure HIV-1 replication fitness in cell culture; however, there is no agreement regarding which assays best correlate with clinical outcomes. A major limitation is that there is no high-throughput assay that incorporates an internal reference strain as a control and utilizes intact virus isolates. Some retrospective studies have demonstrated statistically significant correlations between HIV-1 replication fitness and clinical outcomes in some patient populations. However, different studies disagree as to which clinical outcomes are most closely associated with fitness. This may be in part due to assay design, sample size limitations, and differences in patient populations. In addition, the strength of the correlations between fitness and clinical outcomes is modest, suggesting that, at present, it would be difficult to utilize these assays for clinical management.     

Herpes simplex virus infection induces replication of human immunodeficiency virus type 1

Genital herpes has been associated with increased efficiency of the sexual transmission and enhanced replication of human immunodeficiency virus type 1 (HIV-1). In this study we demonstrate that exposure to infectious or heat-inactivated herpes simplex virus (HSV) type 1 or 2 virions increases HIV-1 expression in macrophages at least in part by inducing NF-kappaB activity. Neutralizing antibodies to the HSV glycoprotein gB or gD markedly attenuated these virion-mediated effects on HIV-1 expression in macrophages. Thus HSV infection of macrophages that reside in genital mucosal tissue induces HIV-1 replication in these cells. Our study may have implications for the management of patients who are coinfected with the two viruses.     

Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor

The current model for HIV-1 envelope-coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell-cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell-cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope-coreceptor interaction.     

Human immunodeficiency virus type 1 tropism for human macrophages.

Human immunodeficiency virus (HIV) infects cells of the monocyte/macrophage lineage in addition to lymphocytes, and infection of these cells may be responsible for viral persistence and dissemination, encephalopathy of the acquired immunodeficiency syndrome and other sequelae of HIV infection. We have developed an in vitro model utilizing peripheral-blood monocyte-derived macrophages to study HIV-1 infection of macrophages. HIV-1 isolates vary greatly in their ability to infect and replicate in macrophages, from highly restricted to highly productive infection. Productively infected macrophages undergo syncytium formation but remain viable in culture and support sustained levels of virus production for prolonged periods. Transformed monocytoid and lymphoid cell lines, however, show very different patterns of permissiveness for HIV-1 strains and do not reflect their corresponding primary cell types in studies of host cell tropism. Studies on viral entry show that the CD4 molecule, known to be the HIV receptor on lymphoid cells, is expressed at low levels on the surface of macrophages as well, where it functions as the receptor for viral entry. Therefore, differential host cell tropism does not result from the use of an alternative macrophage-specific receptor instead of CD4.     

Inheritance pattern of mutant human immunodeficiency virus type 1 coreceptor gene CCR5 in an Indian family

The most common form of mutation found in the CCR5 gene has been the precise 32-base pair (bp) deletion in the region corresponding to second extracellular loop of the chemokine receptor CCR5. Individuals homozygous for the delta 32 allele of CCR5 usually remain uninfected despite multiple exposures to HIV, whereas heterozygous individuals support less virus replication and show slower progression of the disease. This mutant allele in either homozygous or heterozygous form is quite common in white people of European heritage. Earlier work involving large populations of Asians and Africans failed to detect the presence of this mutant allele. We screened 145 normal unrelated healthy Indians and found one person who was heterozygous for the delta 32 allele of CCR5. We studied the inheritance of this deleted allele in this person's family. One parent, one of two sons, and the only daughter possessed this mutant allele. We cloned the entire coding region of wild-type and mutant alleles of CCR5 gene from the heterozygous individual mentioned and studied its coreceptor functions. The mutant allele had only a moderate interfering effect on coreceptor activity of the wild-type CCR5 allele in a cell fusion assay. We also report an improved method of genotyping CCR5 gene in this communication.     

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.     

Population-based sequencing of the V3 region of env for predicting the coreceptor usage of human immunodeficiency virus type 1 quasispecies

Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.     

Replicative fitness of CCR5-using and CXCR4-using human immunodeficiency virus type 1 biological clones

CCR5-tropic viruses cause the vast majority of new HIV-1 infections while about half of the individuals infected with HIV-1 manifest a co-receptor switch (CCR5 (R5) to CXCR4 (X4)) prior to accelerated disease progression. The underlying biological mechanisms of X4 outgrowth in AIDS patients are still poorly understood. Although X4 viruses have been associated with increased "virulence" in vivo, in vitro replication and cytopathicity studies of X4 and R5 viruses have led to conflicting conclusions. We studied the replicative fitness of HIV-1 biological clones with different co-receptor tropism, isolated from four AIDS patients. On average, R5 and X4 clones replicated equally well in mitogen-activated T cells. In contrast, X4 variants were transferred more efficiently from dendritic cells to autologous CD4+ T cells. These observations suggest that interaction between X4 viruses, DC and T cells might contribute to the preferential outgrowth of X4 viruses in AIDS patients.     

HIV-1包膜V3区在病毒侵入靶细胞中的增强作用

目的　明确Ⅰ型人免疫缺陷病毒 (humanimmunodeficiencyvirustype 1,HIV 1)包膜糖蛋白gp12 0第三可变区 (variableregion ,V3)在病毒进入靶细胞的早期阶段中的作用。方法　合成了 3个环状V3多肽 ,包括V3 BH10、V3 ADA和V3 89.6以及直链和短链多肽V3 BH10 /CA、V3 NNT2 4和V3 IRI12。构建了 3株分别带HIVHXB2株、ADA株和 89.6株包膜的伪病毒并观察了多肽对不同嗜性HIV侵入靶细胞能力的影响。结果　 (1)HIV 1V3区多肽以毒株特异性模式增强HXB2、ADA和 89.6进入靶细胞 ;(2 )直链V3多肽与其环状多肽具有相似作用 ;带有C末端的短肽V3 NNT2 4也有与长链V3 BH10 /CA相近的作用 ,只有中央保守区的短链V3 IRI12没有类似功能。结论　本研究首次发现HIV 1V3区在病毒进入靶细胞早期阶段中具有毒株特异性增强病毒侵入的作用。该发现有助于进一步明确HIV 1进入靶细胞的机制     

Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determines the coreceptor usage for CXCR4 and CCR5.

HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.     

Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR.

Human immunodeficiency virus type 1 (HIV-1) phenotype variability plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected individuals is associated with a rapid decline of CD4+ T cells, rapid disease progression, and reduced survival time after AIDS diagnosis. The strong association between the SI capacity of HIV-1 and the presence of positively charged amino acid residues at positions 306 and/or 320 in the third variable domain (V3) of gp120 could here be confirmed in 97% of 402 primary HIV-1 isolates, indicating that the V3 genotype may be useful for prediction of the viral phenotype. The V3 DNA sequences revealed a remarkably limited codon usage for the amino acid residues that are responsible for virus phenotype. On the basis of this limited SI-specific DNA sequence variation, four SI-specific oligonucleotides were designed for selective amplification of V3 from SI but not non-SI HIV-1 isolates. This PCR analysis allowed the prediction of the biological phenotype of HIV-1 isolates on the basis of the V3 genotype and may prove to be useful for monitoring SI capacity of HIV-1 isolates in infected individuals.     

Deficient dimerization of human immunodeficiency virus type 1 RNA caused by mutations of the u5 RNA sequences

The human immunodeficiency virus type 1 (HIV-1) virion contains two copies of genomic RNA that are noncovalently attached along a region at their 5' ends, in which two contact sites have been observed by electron microscopy. One of these sites is believed to be the stem-loop 1 (SL1) sequence which serves as the dimerization initiation site (DIS), and the other site, closer to the 5' end of the viral RNA, may involve the R or U5 RNA sequences. In this study, we present biochemical evidence showing that alteration of the U5 RNA sequence in the context of full-length viral RNA leads to diminished dimerization of virion RNA. In particular, two stretches of GU-rich sequences, which are located at nucleotides (nt) 99 to 108 and nt 112 to 123 within U5, were either deleted or substituted with exogenous sequences. The mutated viruses thus generated all exhibited deficient RNA dimerization. This dimerization deficit was not corrected by second-site mutations that preserved local RNA structures, such as the poly(A) hairpin, and was overcome to only a limited extent by compensatory mutations within Gag; these mutations were identified after long-term culture of the relevant mutant viruses in permissive cell lines and were able to restore viral infectiousness and RNA packaging to wild-type levels. Therefore, these GU sequences do not regulate RNA dimerization by the formation of local secondary structures nor by the maintenance of efficient viral RNA packaging; instead, they may mediate direct RNA-RNA interactions in the dimer structure. In contrast, mutation of palindrome 5'-AAGCUU-3', which resides within R and crowns the poly(A) hairpin, did not affect either RNA dimerization or RNA packaging.     

Inhibition of human immunodeficiency virus type 1 replication by nonionic block polymer surfactants

Eight block copolymers of hydrophilic polyoxy-ethylene and hydrophobic polyoxypropylene were examined for their effects on the replication of human immunodeficiency virus type 1 (HIV-1) in H9 cells. Although the polymers decreased cellular replication, they did not appear to be toxic to the cells; rather, they seemed to arrest cellular growth. Three triblock copolymers were found to inhibit HIV replication at low concentrations. Maximum inhibition was achieved at concentrations of 50 μUg/ml by day 5 following infection. The combination of azidothymidine with both HIV-1-inhibitory and noninhibitory copolymers resulted in antagonistic effects, with an increase in viral replication, compared to treatment with copolymers or azidothymidine alone. These copolymers should be useful in the study of the mechanism of HIV replication in cell cultures and may yield clinically useful compounds in combination therapies for HIV infection. © 1994 Wiley-Liss, Inc.     

The nontoxic tripeptide glycyl-prolyl-glycine amide inhibits the replication of human immunodeficiency virus type 1

OBJECTIVE: To determine whether short peptides corresponding to the RGPGR motif of the V3 loop of gp 120 have anti-human immunodeficiency virus type 1 (anti-HIV-1) activity. DESIGN/METHODS: Short peptides were tested against the HIV-1 laboratory strains and clinical isolates. RESULTS: The tripeptide glycyl-prolyl-glycine amide (GPG-NH2) inhibited the replication of both laboratory strains and 47 clinical isolates, including 19 strains that were resistant to other drugs or that were from patients with failing therapy. The 50% inhibitory concentrations values were 2.7 to 37 microM. Phenotypic change of two isolates from nonsyncytia-inducing to syncytia-inducing did not change their sensitivity to GPG-NH2. The tripeptide added to the antiviral effect of both zidovudine and ritonavir. CONCLUSIONS: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action. Glycyl-prolyl-glycine-NH2 might prove useful by itself or as a lead compound for the treatment of drug-resistant HIV-1. Glycyl-prolyl-glycine-NH2 is currently undergoing phase I/II human clinical trials in Sweden.     

Virus-cell membrane fusion does not predict efficient infection of alveolar macrophages by human immunodeficiency virus type 1 (HIV-1).

Alveolar macrophages (AM) are the principal target cells for HIV-1 in lung tissue. To investigate the mechanisms of HIV-1 infection and efficient replication in these cells we isolated AM from 14 HIV-1 negative donors and exposed them to two virus isolates, either N1T, which replicates well in T lymphocytic and monocytic cell lines, or ADA, a monocytotropic virus. Membrane fluorescence dequenching assays demonstrated that HIV-1/N1T fuses efficiently with AM plasma membranes at neutral pH and that this interaction requires cellular CD4. Despite efficient fusion, AM from eight of 14 donors were not susceptible to productive infection with N1T. In contrast, ADA replicated in all AM populations tested. Soluble CD4 blocked infection of AM by either N1T or ADA, indicating that, like membrane fusion, entry of infectious virus requires an interaction with cellular CD4. Analysis of HIV-1 DNA accumulation in infected cells by enzymatic amplification revealed that productive infection by ADA correlated with a high HIV-1 DNA copy number and abortive infection by N1T was characterized by little or no stable cDNA. These studies suggest that the differences between the two HIV-1 strains studied in their ability to replicate in AM reside in phases of the virus life cycle that follow virus-cell fusion.     

The intensity of immune activation is linked to the level of CCR5 expression in human immunodeficiency virus type 1-infected persons

Immune activation is a main driver of AIDS- and non-AIDS-linked morbidities in the course of HIV-1 infection. As CCR5, the main HIV-1 co-receptor, is not only a chemokine receptor but also a co-activation molecule expressed at the surface of T cells, it could be directly involved in this immune activation. To test this hypothesis, we measured by flow cytometry the mean number of CCR5 molecules at the surface of non-activated CD4⁺ T cells (CCR5 density), which determines the intensity of CCR5 signalling, and the percentage of CD8⁺ T cells over-expressing CD38 (CD38 expression), a major marker of immune activation, in the blood of 67 HIV-1-infected, non-treated individuals. CCR5 density was correlated with CD38 expression independently of viral load (P = 0·016). CCR5 density remained unchanged after highly active anti-retroviral therapy (HAART) introduction or cessation, whereas CD38 expression decreased and increased, respectively. Moreover, pre-therapeutic CCR5 density was highly predictive (r = 0·736, P < 10⁻⁴) of residual CD38 over-expression after 9 months of HAART. Hence, CCR5 might play an immunological role in HIV-1 infection as a driver of immune activation. This could explain why CCR5 antagonists may have an inhibitory effect on immune activation.