Replication of HIV-1 in primary monocyte cultures

A subpopulation of human monocytes began to express surface CD4 during its in vitro cultivation in the presence of recombinant M-CSF and became susceptible to infection with an isolate of HIV-1 [AD-87(M)], originally recovered from a seropositive individual by cocultivation with primary monocytes. Approximately 10% of the adherent cells synthesized CD4; a similar fraction was infectable with AD-87(M) and produced high levels of progeny particles. An interesting feature of the HIV-1 infection of primary monocytes was the detection of gp 160/120 inside but not on the surface of virus-producing cells. The implication of these results on HIV infection in vivo will be discussed.     

Replication of HIV-1 in Vivo and in Vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

Replication of HIV-1 and HIV-2 in human bone marrow cultures.

The bone marrow is a target organ for the human immunodeficiency virus (HIV), but the mechanisms by which suppression of hematopoiesis occurs during the course of HIV infection are not well understood. To study this issue, the effect of several different HIV-1 isolates (monotrophic and lymphotrophic) and one HIV-2 isolate on in vitro colony formation by BFU-E and CFU-GM from normal human marrow were examined. The monotrophic strain AD-87 (M) failed to replicate in marrow cultures as documented by RT, and colony formation by BFU-E and CFU-GM was unaffected by this virus. A derivative of this isolate AD-87 (M-P), which was replicated in peripheral blood lymphocytes (PBL), however, replicated well and markedly inhibited colony formation by BFU-E and CFU-GM. Two additional PBL isolates replicated less efficiently; neither inhibited CFU-GM but one consistently inhibited BFU-E colony formation. Inhibition of colony formation by the HIV-1 isolates was a late event, presumably a secondary lysis of cells, since up to 7 days after inoculation colony numbers were normal but diminished markedly by 10 days, and since only up to 10% of the cells of the monocyte lineage contained detectable virus by in situ, EM, and IFA studies. In contrast, the HIV-2 isolate was so lytic that by 4 days after inoculation the majority of the marrow cells were destroyed.     

Alterations of the B-Cell Response by HIV-1 Replication

While the hallmark of HIV-1 infection is the progressive depletion of CD4(+) T cells, extensive B-cell dysfunction ensues that impairs the quality of the humoral response. HIV-1 infection causes hypergammaglobulinemia, polyclonal activation, loss of memory B-cell subsets, B-cell exhaustion, aberrant B-cell surface markers, and impaired humoral responses against infections and vaccinations. The totality of the mechanisms that contribute to B-cell dysfunction in vivo is unknown, although roles for HIV proteins (Env, Tat, and Nef) and virions binding to CD21 on B cells have been identified. Recent studies suggest that early antiretroviral therapy, that minimizes virus replication, can profoundly preserve the early B-cell response to HIV-1. Thus, it is clear that there is an intricate interplay between HIV replication and stimulation of the host B-cell response to infection. A better understanding of how HIV-1 subverts a productive B-cell response is needed to inform vaccine strategies that aim to elicit long-lived plasma cells and memory B-cell responses that can act quickly upon antigen stimulation.     

Replication of HIV-1 in vivo and in vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

Erratum to: Alterations of the B-Cell Response by HIV-1 Replication

While the hallmark of HIV-1 infection is the progressive depletion of CD4⁺ T cells, extensive B-cell dysfunction ensues that impairs the quality of the humoral response. HIV-1 infection causes hypergammaglobulinemia, polyclonal activation, loss of memory B-cell subsets, B-cell exhaustion, aberrant B-cell surface markers, and impaired humoral responses against infections and vaccinations. The totality of the mechanisms that contribute to B-cell dysfunction in vivo is unknown, although roles for HIV proteins (Env, Tat, and Nef) and virions binding to CD21 on B cells have been identified. Recent studies suggest that early antiretroviral therapy, that minimizes virus replication, can profoundly preserve the early B-cell response to HIV-1. Thus, it is clear that there is an intricate interplay between HIV replication and stimulation of the host B-cell response to infection. A better understanding of how HIV-1 subverts a productive B-cell response is needed to inform vaccine strategies that aim to elicit long-lived plasma cells and memory B-cell responses that can act quickly upon antigen stimulation.     

Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells

Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication.     

Conservation of a Hairpin Ribozyme Sequence in HIV-1 Is Required for Efficient Viral Replication

We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MN_Ahas a substitution of A for G immediately 3′ to the cleavage site and HIV-1 MN_GChas two substitutions in the flanking sequences that are complementary to the ribozyme.In vitrostudies confirmed that neither the MN_A- nor the MN_GC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MN_GC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MN_Aand MN_GCmutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MN_Aor partial reversion of MN_GCto wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.     

HIV-1-Specific CD4+ T Cell Responses in Chronically HIV-1 Infected Blippers on Antiretroviral Therapy in Relation to Viral Replication Following Treatment Interruption

The impact of transient viral load blips on anti-HIV-1 immune responses and on HIV-1 rebound following treatment interruption (TI) is not known. Clinical and immunological parameters were measured during 40 weeks of antiretroviral therapy (ART) and following TI in an observational cohort of 16 chronically HIV-1-infected subjects with or without observed viral load blips during ART. During therapy, blips in seven subjects were associated with higher anti-HIV-1 (p24) CD4⁺ T cell lymphoproliferative responses (p = 0.04), without a significant difference in T cell activation or total anti-HIV-1 CD8⁺ T cell interferon-γ (IFN-γ) responses when compared to nine matched non-blippers. Therapy interruption resulted in a significantly higher viral rebound in blippers by 8 week despite retention of higher lymphoproliferative p24 responses (p = 0.01) and a rise in CD3⁺ T cell activation (p = 0.04) and anti-HIV-1 CD8⁺ T cell responses in blippers by week 4 when compared to non-blippers. Past week 4 of interruption, therapy re-initiation criteria were also met by a higher frequency in blippers by week 14 (p < 0.04) with no difference between groups by week 24. These data support that blippers have higher anti-HIV lymphoproliferative responses while on ART but experience equal to higher viral rebound as compared to matched non-blippers upon TI.     

HIV-1 Prevention for HIV-1 Serodiscordant Couples

A substantial proportion of HIV-1 infected individuals in sub-Saharan Africa are in stable relationships with HIV-1 uninfected partners, and HIV-1 serodiscordant couples thus represent an important target population for HIV-1 prevention. Couple-based HIV-1 testing and counseling facilitates identification of HIV-1 serodiscordant couples, counseling about risk reduction, and referrals to HIV-1 treatment, reproductive health services, and support services. Maximizing HIV-1 prevention for HIV-1 serodiscordant couples requires a combination of strategies, including counseling about condoms, sexual risk, fertility, contraception, and the clinical and prevention benefits of antiretroviral therapy (ART) for the HIV-1-infected partner; provision of clinical care and ART for the HIV-1-infected partner; antenatal care and services to prevent mother-to-child transmission for HIV-1-infected pregnant women; male circumcision for HIV-1-uninfected men; and, pending guidelines and demonstration projects, oral pre-exposure prophylaxis (PrEP) for HIV-1-uninfected partners.     

HIV-1 superinfection

During the past year, a number of reports have described HIV-1 superinfection in human subjects, defined as the reinfection of an individual with a second heterologous strain of HIV-1. These reports have challenged the assumption that HIV-1-specific immune responses generated during primary infection are protective against subsequent infection and have raised concern, not only with respect to HIV-1-positive individuals engaging in unsafe sex but also from the standpoint of developing effective vaccines. Herein we review the published reports of HIV-1 superinfection and highlight studies providing additional insight into the potential for HIV-1 superinfections to affect the global epidemic.     

NucliSens HIV-1 QT和Amplicor HIV-1 monitor1.5方法测定HIV-1病毒载量的比较研究

目的进行NuchSens HIV-1 QT和Amplicor HIV-1 monitor 1．5检测相同临床样品HIV-1病毒载量值之间的比较研究。方法收集临床样本82份，使用两种方法测定病毒载量，对病毒载量值进行统计分析。结果未测到核酸的和两种方法病毒载量对数值之差小于0．5的占88．9％；用△log10 VL〈0．5的56份样本统计两种方法的相关性，相关系数为0．956。结论NucliSens HIV-1 QT和Amphcor HIV-1 monitor 1．5两种方法测定的HIV病毒载量值有很好的相关性。     

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.     

Genital HIV-1 RNA predicts risk of heterosexual HIV-1 transmission

High plasma HIV-1 RNA concentrations are associated with an increased risk of HIV-1 transmission. Although plasma and genital HIV-1 RNA concentrations are correlated, no study has evaluated the relationship between genital HIV-1 RNA and the risk of heterosexual HIV-1 transmission. In a prospective study of 2521 African HIV-1 serodiscordant couples, we assessed genital HIV-1 RNA quantity and HIV-1 transmission risk. HIV-1 transmission linkage was established within the partnership by viral sequence analysis. We tested endocervical samples from 1805 women, including 46 who transmitted HIV-1 to their partner, and semen samples from 716 men, including 32 who transmitted HIV-1 to their partner. There was a correlation between genital and plasma HIV-1 RNA concentrations: For endocervical swabs, Spearman's rank correlation coefficient ρ was 0.56, and for semen, ρ was 0.55. Each 1.0 log(10) increase in genital HIV-1 RNA was associated with a 2.20-fold (for endocervical swabs: 95% confidence interval, 1.60 to 3.04) and a 1.79-fold (for semen: 95% confidence interval, 1.30 to 2.47) increased risk of HIV-1 transmission. Genital HIV-1 RNA independently predicted HIV-1 transmission risk after adjusting for plasma HIV-1 quantity (hazard ratio, 1.67 for endocervical swabs and 1.68 for semen). Seven female-to-male and four male-to-female HIV-1 transmissions (incidence <1% per year) occurred from persons with undetectable genital HIV-1 RNA, but in all 11 cases, plasma HIV-1 RNA was detected. Thus, higher genital HIV-1 RNA concentrations are associated with greater risk of heterosexual HIV-1 transmission, and this effect was independent of plasma HIV-1 concentrations. These data suggest that HIV-1 RNA in genital secretions could be used as a marker of HIV-1 sexual transmission risk.     

Mu-opioid modulation of HIV-1 coreceptor expression and HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (i.v.DUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the mu-opioid receptor. Our results show that DAMGO, a selective mu-opioid agonist, increases CXCR4 and CCR5 expression in both CD3(+) lymphoblasts and CD14(+) monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the mu-opioid receptor based on the ability of a mu-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of mu-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.     

Salivary detection of HIV-1 antibodies using recombinant HIV-1 peptides

Salivary antibodies may play a role in the absence of HIV-1 transmission by saliva. We evaluated the presence of salivary IgG antibodies to HIV-1 using a recombinant ELISA. Whole saliva was collected from 21 HIV-1-seropositive individuals and assayed in an ELISA, ASQ (Beckman Instruments, Brea, CA), consisting of a panel of six HIV-1 recombinant peptides. Saliva samples from 20 individuals demonstrated IgG to one or more peptides and 18 to two or more peptides. Samples from 20 seropositive individuals were reactive with the gp41 peptide, whereas only 12 were reactive with the two gp120 peptides. Nineteen of twenty salivas also had detectable IgG antibodies to HIV-1 by Western blotting. The results indicate that viral-specific IgG antibodies are present in the saliva of a high percentage of HIV-infected individuals and that a recombinant peptide ELISA for saliva might be useful for the detection of HIV-1 infection.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.