Differential tropism of clinical HIV-1 isolates for primary monocytes and promonocytic cell lines.

Previously we demonstrated a correlation between a nonsyncytium-inducing (NSI), non-T-cell line tropic phenotype of HIV-1 isolates and the capacity to replicate in primary monocyte-derived macrophages (MDM). Here we demonstrate that these NSI, monocytotropic HIV-1 isolates lack the capacity to replicate in two promonocytic cell-lines, HL60 and U937. In contrast, most syncytium-inducing (SI) HIV-1 isolates with tropism for T-cell lines and generally non-monocytotropic were able to establish a productive infection in promonocytic cell lines. Similar differences in tropism for monocytes and promonocytic cell lines were observed with infectious molecular clones. Our results indicate that virological studies on promonocytic cell lines do not necessarily pertain to the HIV-1 infection of monocytes in vivo.     

Macrophage tropism of HIV-1

Both in vivo and in vitro studies clearly demonstrate that cells of the mononuclear phagocyte lineage are major hosts for human immunodeficiency virus type 1 (HIV-1) replication. Presumably these cells play a key role in the pathogenesis of acquired immunodeficiency syndrome (AIDS). To further delineate the interactions between HIV-1 and host cells, the susceptibility and permissivity of normal human peripheral blood-derived monocyte/macrophages (M/M) and T lymphocytes, and neoplastic monocytoid and lymphoid cell lines to various HIV-1 isolates was assessed. The results suggest: (1) "fresh" isolates recovered from patients and propagated only in normal host cells exhibit a dual tropism for both M/M and T cells, regardless of their tissue of origin or the cell type from which they were isolated; (2) the repeated passage of an HIV-1 isolate through normal M/M does not generally result in the loss of the ability to infect normal T cells nor vice versa; (3) the majority of fresh HIV-1 isolates do not infect neoplastic cells of either origin, and those that do show no preference for monocytoid or lymphoid targets, regardless of their cell origin.     

Relative activities of HIV-1-IIIB and HIV-1BaL LTR and tat in primary monocytes and lymphocytes

The mechanisms determining the ability of some but not other strains of human immunodeficiency virus type 1 (HIV-1) to grow in peripheral blood monocyte-macrophages are presently unclear. The tat gene of HIV-1-IIIB which replicates poorly in human macrophages, and the tat gene of HIV-1-BaL, which replicates to high titers in the same cells in transient expression systems with their respective long terminal repeats (LTR) driving a reporter chloramphenicol acetyl transferase (CAT) gene were compared. The authors hypothesized that the tat gene and LTR of BaL might help account for its efficient growth in primary monocyte-macrophages by virtue of a high activity in these cells relative to that of the IIIB tat and LTR. Primary peripheral blood lymphocytes and monocytes were cotransfected with either the HIV-1BaL or HIV-1-IIIB LTR fused to the CAT gene and their respective tat genes. The IIIB tat and LTR were at least as active in primary lymphocytes as the BaL combination, and both tat-LTR pairs were more active in primary lymphocytes than monocytes. The same relative activities were also observed in primary monocytes after in vitro maturation to macrophages prior to transfection. These data strongly suggest that neither the tat gene nor the LTR of HIV-1-IIIB and HIV-1BaL can account for the great ability of the latter or the inability of the former to grow in monocyte-macrophages.     

Viral determinants of HIV-1 macrophage tropism

Macrophages are important target cells for HIV-1 infection that play significant roles in the maintenance of viral reservoirs and other aspects of pathogenesis. Understanding the determinants of HIV-1 tropism for macrophages will inform HIV-1 control and eradication strategies. Tropism for macrophages is both qualitative (infection or not) and quantitative (replication capacity). For example many R5 HIV-1 isolates cannot infect macrophages, but for those that can the macrophage replication capacity can vary by up to 1000-fold. Some X4 viruses are also capable of replication in macrophages, indicating that cellular tropism is partially independent of co-receptor preference. Preliminary data obtained with a small number of transmitted/founder viruses indicate inefficient macrophage infection, whereas isolates from later in disease are more frequently tropic for macrophages. Thus tropism may evolve over time, and more macrophage tropic viruses may be implicated in the pathogenesis of advanced HIV-1 infection. Compartmentalization of macrophage-tropic brain-derived envelope glycoproteins (Envs), and non-macrophage tropic non-neural tissue-derived Envs points to adaptation of HIV-1 quasi-species in distinct tissue microenvironments. Mutations within and adjacent to the Env-CD4 binding site have been identified that determine macrophage tropism at the entry level, but post-entry molecular determinants of macrophage replication capacity involving HIV-1 accessory proteins need further definition.     

Dual tropism of HIV-1 envelopes derived from renal tubular epithelial cells of patients with HIV-associated nephropathy

The phenotype of HIV-1 gp120 envelope derived from renal epithelium and peripheral blood mononuclear cells (PBMC) of patients with HIV-associated nephropathy was investigated in vitro. Chimeric viruses were derived from kidney or blood and used to infect primary CD4+T cells, cell lines expressing single co-receptors and a renal epithelial cell line HPT-1. HIV-1 variants derived from renal epithelium were dual tropic whereas simultaneously derived viruses from PBMC were R5-tropic. Utilization of alternative co-receptors CCR3, BONZO and BOB, also differed.     

HIV-1 tropism and survival in vertically infected Ugandan infants

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) may utilize the CXCR4 coreceptor (X4 virus), the CCR5 coreceptor (R5 virus), or both (dual/mixed [DM] virus). We analyzed HIV-1 coreceptor tropism in Ugandan infants enrolled in the HIVNET (HIV Network for Prevention Trials) 012 trial. METHODS: Plasma or serum was analyzed using a commercial coreceptor tropism assay. HIV env subtype was determined by phylogenetic methods. RESULTS: Tropism results were obtained for 57 samples from infants collected 6-14 weeks after birth. Fifty-two infants had only R5 virus, and 5 had either X4 or DM virus. The mothers of those 5 infants also had X4 or DM virus. In infants, subtype D infection was associated with high-level infectivity in CCR5-bearing cells and also with the detection of X4 or DM strains. High-level infectivity in CCR5-bearing cells was associated with decreased infant survival, but infection with X4 or DM virus was not. HIV clones from infants with DM viral populations showed different patterns of coreceptor use. V3 loop sequence-based algorithms predicted the tropism of some, but not all, env clones. CONCLUSIONS: Complex patterns of HIV tropism were found in HIV-infected newborn infants. Subtype D infection was associated with X4 virus and with high-level replication in CCR5-bearing cells. High-level replication of R5 virus was associated with decreased infant survival.     

HAART治疗前后HIV-1细胞嗜性分析

目的 分析7例患者在高效抗逆转录病毒治疗(HAART)前和治疗后,艾滋病病毒Ⅰ型(HIV-1)细胞嗜性的改变及影响因素. 方法 采用BD公司的流式细胞仪,通过绝对计数法测定CD 4 T淋巴细胞计数和CD 8 T淋巴细胞计数,荧光标记物为BD TriTEST CD3FITC/CD4PE/CD45PerCP.采用核酸序列扩增实验(NASBA)测定病毒载量(生物梅里埃公司提供仪器和试剂).套式聚合酶链反应(Nested-PCR)对gp120V3环区基因进行扩增、测序分析,并确定HIV-1细胞嗜性. 结果 在7例患者中,有3例发生细胞嗜性转换,2例是从R5到X4嗜性的转换,1例是X4到R5嗜性的转换.在7例患者中,病毒细胞嗜性转换与患者病毒载量、CD 4、CD 8 T淋巴细胞之间无相关性. 结论 HIV-1细胞嗜性的转换与疾病进展的关系不确定;HAART治疗对嗜性转换的作用还不明确.     

Comparison of two genotypic algorithms to determine HIV-1 tropism

Objectives

One or both of two co‐receptors, CCR5 (R5) and CXCR4 (X4), are used by HIV‐1 to enter into host cells. The glycoprotein 120 (gp120) V3 sequence is correlated with the R5 and X4 phenotype. CCR5 inhibitors are specifically active against R5 viruses, suggesting the need to determine tropism before the use of these antagonists. A comparison of the position‐specific scoring matrices (PSSM) and Geno2pheno algorithms based on the V3 loop gp120 sequences and previously described to be correlated to the R5 or X4 phenotype was carried out.

Methods

V3 envelope (env) genes from 83 plasma samples were amplified and sequenced, and 69 sequences were analysed with the PSSM and Geno2pheno algorithms.

Results

These two algorithms were concordant in 86.5% of cases. The Geno2pheno algorithm gave a tropism result more frequently than the PSSM algorithm, but R5X4 or X4 viruses were less frequently detected by the Geno2pheno algorithm. R5X4 or X4 tropism was predicted in 29.9% of samples. There was more R5X4 co‐receptor use in the antiretroviral‐treated group than in the antiretroviral‐naïve group.

Conclusions

It is advisable to run a validated co‐receptor use prediction tool before using co‐receptor antagonists. If genotyping methods are considered, the PSSM and Geno2pheno algorithms are complementary and both are necessary. The association between predicted co‐receptor use and virological response to co‐receptor antagonists needs to be thoroughly evaluated.     

European guidelines on the clinical management of HIV-1 tropism testing

Viral tropism is the ability of viruses to enter and infect specific host cells and is based on the ability of viruses to bind to receptors on those cells. Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. In most European countries, HIV tropism is identified with tropism phenotype testing. New data support genotype analysis of the HIV third hypervariable loop (V3) for the identification of tropism. The European Consensus Group on clinical management of tropism testing was established to make recommendations to clinicians and clinical virologists. The panel recommends HIV-tropism testing for the following groups: drug-naive patients in whom toxic effects are anticipated or for whom few treatment options are available; patients who have poor tolerability to or toxic effects from current treatment or who have CNS pathology; and patients for whom therapy has failed and a change in treatment is considered. In general, an enhanced sensitivity Trofile assay and V3 population genotyping are the recommended methods. Genotypic methods are anticipated to be used more frequently in the clinical setting because of their greater accessibility, lower cost, and faster turnaround time than other methods. For the interpretation of V3 loop genotyping, clinically validated systems should be used when possible. Laboratories doing HIV tropism tests should have adequate quality assurance measures. Similarly, close collaboration between HIV clinicians and virologists is needed to ensure adequate diagnostic and treatment decisions.     

Human topoisomerase I promotes HIV-1 proviral DNA synthesis: implications for the species specificity and cellular tropism of HIV-1 infection

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10-15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.     

Antiretroviral drug resistance, HIV-1 tropism, and HIV-1 subtype among men who have sex with men with recent HIV-1 infection

OBJECTIVE: Antiretroviral drug treatment may be complicated in individuals infected with antiretroviral drug-resistant or non-subtype B HIV-1 strains. HIV-1 tropism may also affect disease progression. We analyzed antiretroviral drug resistance, HIV-1 subtype, and HIV-1 tropism among 195 men who have sex with men from six major cities in the United States, using samples collected within 6 months of HIV-1 seroconversion (1999-2003). METHODS: HIV-1 genotyping was performed using the ViroSeq HIV-1 Genotyping System. HIV-1 tropism was determined using a commercial assay. HIV-1 subtyping was performed by phylogenetic analysis of pol region sequences. RESULTS: Thirty-one (15.9%) of the men had evidence of antiretroviral drug resistance. Seven (3.6%) men had multi-class resistance, including three (1.5%) with resistance to all three antiretroviral drug classes. We found no statistically significant association of antiretroviral drug resistance with demographic factors, sexual practices, self-reported sexually transmitted infections, use of recreational drugs, or use of antiretroviral drug post-exposure prophylaxis. All samples were HIV-1 subtype B. Four men had CXCR4-using HIV-1 strains. One man with a CXCR4-using strain also had antiretroviral drug resistance. CONCLUSIONS: Antiretroviral drug resistance is relatively common among recently infected men who have sex with men in the United States. CXCR4-using strains were detected in a small number of these infections, which were all subtype B HIV-1.     

Antibodies and complement enhance binding and uptake of HIV-1 by human monocytes.

OBJECTIVE: To characterize antibody- and complement-mediated binding and uptake of HIV-1 by human monocytes. DESIGN: The first step in the infection of the monocyte by HIV-1 is binding of the virus to the susceptible cell. Procedures were designed to assess the influence of anti-HIV-1 antibodies and complement on this binding, and to study the process of internalization following binding. METHODS: Human monocytes were incubated with fluorescein-labelled purified HTLV-IIIB virions and human sera with high-titre anti-HIV-1 antibodies and/or complement. Binding and uptake of virus by the monocytes was measured as fluorescence per cell by flow cytometry. RESULTS: Binding of purified HIV-1 to monocytes was increased by complement and, to a lesser extent, by anti-HIV-1 antibodies. Uptake of HIV-1 bound to the monocyte appeared to be mediated by antibodies and was increased further by the presence of complement. Complement alone, however, resulted in the uptake of only a small part of the bound virus. CONCLUSIONS: Complement significantly increases the binding of HIV-1 to human monocytes, and a combination of antibodies and complement efficiently mediates uptake of HIV-1 by monocytes.     

Function of lymphocytes and monocytes in silicosis.

The function of lymphocytes and monocytes was compared in a group of 18 ex-sandblasters with silicosis and 19 control subjects. Previously noted depressed lymphocytic proliferation in response to low concentrations of concanavalin A (ConA) in subjects with silicosis was not due to factors in the serum nor correctable by supplementation with allogeneic lymphocytes. Monocytes from the peripheral blood of the subjects with silicosis responded less to a chemotactic stimulus (zymosan-activated serum) than did monocytes from normal laboratory personnel; however, there was no difference in monocytic chemotaxis when groups of similar age were compared. There was a significant correlation between age and the monocytic response to chemotactic stimuli in the studied population (laboratory personnel, silicotic subjects, and age-matched control subjects). Overall, our data suggest subtle effects of silica on selected T-lymphocytic populations involved with responsiveness to the mitogen, concanavalin A, but no effects on monocytic function. Monocytic chemotaxis is unaffected by exposure to silica but is inversely related to age.     

HIV-1 Vpu represents a minor target for cytotoxic T lymphocytes in HIV-1-infection

We have previously shown that Vpu is rarely targeted by HIV-1-specific cytotoxic T lymphocytes (CTL). The present report extends these findings and describes the characterization of the first CTL epitope within HIV-1 Vpu, identified in an individual with long-term non-progressive HIV-1 infection. The epitope was shown to be highly conserved among HIV clade B sequences and is restricted by HLA-A*3303, an HLA allele commonly seen in Asian and west-African populations.     

Cell-associated HIV-1 messenger RNA and DNA in T-helper cell and monocytes in asymptomatic HIV-1-infected subjects on HAART plus an inactivated HIV-1 immunogen.

OBJECTIVE: We examined the effect of an HIV-1-specific immune-based therapy on cell-associated HIV-1 DNA and RNA. DESIGN: Five HIV-1-infected subjects receiving HIV-1 immunogen plus HAART were compared with three HIV-1-infected subjects who received incomplete Freund's adjuvant (IFA) plus HAART. METHODS: Cell-associated HIV-1 RNA or DNA in lymphocytes and monocytes was determined using a dual immunophenotyping/in situ hybridization assay with or without in situ PCR amplification. RESULTS: Cell-associated HIV-1 RNA in CD4 cells correlated with plasma RNA overall. CD4, HIV-1 gag-pol messenger (m)RNA+ cells decreased in the immunogen plus HAART group compared with the IFA plus HAART group. Decreases in HIV-1 DNA+ CD4 cells were observed in the immunogen plus HAART compared with the IFA plus HAART group. Decreases in HIV-1 gag-pol mRNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group. Consistent with the findings in CD4 cells, decreases in HIV-1 DNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group. CONCLUSIONS: These preliminary observations support the rationale for examining the combination of immune-based therapies and antiretroviral drugs for effective HIV-1 control.