Tetraspanin CD63 is a regulator of HIV-1 replication

Macrophages and CD4⁺ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4⁺ T-cells, dendritic cells, and a CD4⁺ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4⁺ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4⁺ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2).     

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3′ half of the viral genome of subtype A was replaced with the corresponding subtype C3′ half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5′ half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1α-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural β-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES, to CCR5⁺ cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1α, MIP-1β, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by β-chemokines, MIP-1α, MIP-1β, and RANTES as well as the HIV-1 infection.     

Down-regulation of CXCR4 expression by SDF-KDEL in CD34 hematopoietic stem cells: An anti-human immunodeficiency virus strategy

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34⁺ hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34⁺ hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10³ TCID50 HIV-1 infected CD34⁺ hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P < 0.05).     

Mapping of determinants involved in the stimulation of HIV-1 expression by Sam68

Control of HIV-1 RNA processing is central to the replication of the virus. Previously, we demonstrated that the cellular protein Sam68 enhances HIV-1 structural protein expression and RNA 3′ end processing. In this report, we show that Sam68 interacts with unspliced HIV-1 RNA and that other members of the STAR/GSG protein family also promote viral RNA 3′ end processing. We define a portion of the GSG domain (Sam 97-255) as sufficient for enhancement of Rev-dependent expression. In contrast to Sam68, Sam 97-255 increases unspliced RNA processing only in the presence of Rev in 293T cells. In a different cell line, Sam 97-255 enhances HIV-1 gene expression without enhancing RNA 3′ end processing, suggesting that stimulation of 3′ end processing is not required for enhancement of HIV-1 gene expression. Overall, these results indicate that Sam68 and the mutants described affect the composition of the viral RNP to enhance viral protein synthesis.     

The 5′CL-PCBP RNP complex, 3′ poly(A) tail and 2A are required for optimal translation of poliovirus RNA

In this study, we showed that the 5′CL-PCBP complex, 3′ poly(A) tail and viral protein 2A^pro are all required for optimal translation of PV RNA. The 2A^pro-mediated stimulation of translation was observed in the presence or absence of both the 5′CL and the 3′ poly(A) tail. Using protein-RNA tethering, we established that the 5′CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5′CL-PCBP complex and the 3′ poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5′CL and the 3′ poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5′CL-PCBP complex interacts with the 3′ poly(A)-PABP complex to form a 5′-3′ circular complex that facilitates ribosome reloading and stimulates PV RNA translation.     

In vitro replication capacity of HIV-2 variants from long-term aviremic individuals

To establish whether efficient suppression of virus replication in HIV-2-infected individuals is associated with low replicative capacity of HIV-2, replication kinetics of HIV-2 variants from long-term aviremic individuals was analyzed and compared with that of the relatively slow-replicating HIV-1 variants from asymptomatics and long-term nonprogressors (AS/LTNP). On average, HIV-2 from aviremic individuals had lower replication rates than HIV-1 variants from AS/LTNP in cells of 8 donors (0.45 log10 [range 0.14-0.77] vs. 0.58 log10 [range 0.32-0.99] pg RT/ml/day, P = 0.036). The relatively low replication rate of HIV-2 compared to HIV-1 variants was not related to different sensitivities to inhibition by CD8+ T cells or different degrees of infectivity. HIV-2 replication rates increased with progressive infection and with switch from CCR5 to CXCR4 usage. The relatively low replicative capacity of HIV-2 variants from aviremic individuals likely contributes to the low viral load and benign course of infection in these individuals.     

The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes

The phenotypic changes that are induced by immune activation in CD4⁺ T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4⁺ T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4⁺ T lymphocytes.     

High levels of HIV-1 replication show a clear correlation with downmodulation of Bcl-2 protein in peripheral blood lymphocytes of HIV-1-seropositive subjects

Peripheral blood lymphocytes (PBLs) from 51 HIV-1-seropositive subjects with different levels of HIV-1 replication and 20 healthy blood donors were examined for the expression of the antiapoptotic Bcl-2 protein. All the plasma samples from HIV-1 patients were characterized for the presence of HIV-1 p24 and HIV RNA viral load. Bcl-2 protein expression in fresh peripheral blood lymphocytes was studied by different tests, including Western blot and indirect immunofluorescence techniques. Direct immunofluorescence staining, revealed by flow cytometry, was applied to quantify the number of specific anti-Bcl-2 antibody epitope binding sites, thus extrapolating the relative number of Bcl-2 into the cells. The results indicate that the expression of Bcl-2 protein is significantly lower in peripheral blood lymphocytes of HIV-1-seropositive patients showing high levels of viral replication, detected by means of HIV-1 p24 and RNA viral load, with respect to HIV-1 patients with low levels of virus replication and healthy blood donors. The clear-cut inverse correlation between viral replication and Bcl-2 expression reinforces the view that HIV-1-mediated apoptosis probably represents a key mechanism in AIDS pathogenesis. J. Med. Virol. 56:66–73, 1998. © 1998 Wiley-Liss, Inc.     

Selective infection of CD4 effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγ mice

To investigate the events leading to the depletion of CD4⁺ T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34⁺ cells-transplanted NOD/SCID/IL-2Rγ^null mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4⁺ thymocytes and both CD45RA⁺ naïve and CD45RA⁻ memory CD4⁺ T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA⁻ memory CD4⁺ T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T_EM) rather than central memory T lymphocytes. In addition, the majority of p24⁺ cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T_EM in cycling phase and further suggest that the predominant infection in T_EM would lead to the depletion of memory CD4⁺ T lymphocytes in CCR5-tropic HIV-1-infected mice.