Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.     

Restricted 5′-End Gap Repair of HIV-1 Integration Due to Limited Cellular dNTP Concentrations in Human Primary Macrophages

HIV-1 proviral DNA integration into host chromosomal DNA is only partially completed by the viral integrase, leaving two single-stranded DNA gaps with 5′-end mismatched viral DNA flaps. It has been inferred that these gaps are repaired by the cellular DNA repair machinery. Here, we investigated the efficiency of gap repair at integration sites in different HIV-1 target cell types. First, we found that the general gap repair machinery in macrophages was attenuated compared with that in dividing CD4⁺ T cells. In fact, the repair in macrophages was heavily reliant upon host DNA polymerase β (Pol β). Second, we tested whether the poor dNTP availability found in macrophages is responsible for the delayed HIV-1 proviral DNA integration in this cell type because the K_m value of Pol β is much higher than the dNTP concentrations found in macrophages. Indeed, with the use of a modified quantitative AluI PCR assay, we demonstrated that the elevation of cellular dNTP concentrations accelerated DNA gap repair in macrophages at HIV-1 proviral DNA integration sites. Finally, we found that human monocytes, which are resistant to HIV-1 infection, exhibited severely restricted gap repair capacity due not only to the very low levels of dNTPs detected but also to the significantly reduced expression of Pol β. Taken together, these results suggest that the low dNTP concentrations found in macrophages and monocytes can restrict the repair steps necessary for HIV-1 integration.     

Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes

The latency of human immunodeficiency virus type 1 (HIV-1) in resting primary CD4+ T cells is the major barrier for the eradication of the virus in patients on suppressive highly active antiretroviral therapy (HAART). Even with optimal HAART treatment, replication-competent HIV-1 still exists in resting primary CD4+ T cells. Multiple restriction factors that act upon various steps of the viral life cycle could contribute to viral latency. Here we show that cellular microRNAs (miRNAs) potently inhibit HIV-1 production in resting primary CD4+ T cells. We have found that the 3' ends of HIV-1 messenger RNAs are targeted by a cluster of cellular miRNAs including miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in resting CD4+ T cells as compared to activated CD4+ T cells. Specific inhibitors of these miRNAs substantially counteracted their effects on the target mRNAs, measured either as HIV-1 protein translation in resting CD4+ T cells transfected with HIV-1 infectious clones, or as HIV-1 virus production from resting CD4+ T cells isolated from HIV-1-infected individuals on suppressive HAART. Our data indicate that cellular miRNAs are pivotal in HIV-1 latency and suggest that manipulation of cellular miRNAs could be a novel approach for purging the HIV-1 reservoir.     

Evidence for Human Immunodeficiency Virus Type 1 Replication In Vivo in CD14+ Monocytes and Its Potential Role as a Source of Virus in Patients on Highly Active Antiretroviral Therapy

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshly isolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 may replicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productive infection with HIV-1. We investigated HIV-1 replication in CD14(+) monocytes and resting and activated CD4(+) T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 in seven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days during highly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14(+) monocytes as well as in activated and resting CD4(+) T cells throughout the course of study. While significant variation in the decay slopes of HIV-1 DNA was seen among individual patients, viral decay in CD14(+) monocytes was on average slower than that in activated and resting CD4(+) T cells. Measurements of HIV-1 sequence evolution and the concentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, more pronounced in CD14(+) monocytes than in resting CD4(+) T cells. Phylogenetic analyses of HIV-1 sequences indicated that after prolonged HAART, viral populations related or identical to those found only in CD14(+) monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequences in plasma and the three cell populations were identical. CD14(+) monocytes appear to be one of the potential in vivo sources of HIV-1 in patients receiving HAART.     

Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both "activated" and "nonactivated" mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.     

LFA-1 is a key determinant for preferential infection of memory CD4+ T cells by human immunodeficiency virus type 1

Memory CD4+ T cells are considered a stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) and a barrier to eradication of this retroviral infection in patients under therapy. It has been shown that memory CD4+ T cells are preferentially infected with HIV-1, but the exact mechanism(s) responsible for this higher susceptibility remains obscure. Previous findings indicate that incorporation of host-derived intercellular adhesion molecule 1 (ICAM-1) in HIV-1 increases virus infectivity. To measure the putative involvement of virus-anchored ICAM-1 in the preferential infection of memory cells by HIV-1, quiescent and activated naive and memory T-cell subsets were exposed to isogenic virions either lacking or bearing ICAM-1. Memory CD4+ T cells were found to be more susceptible than naive CD4+ T cells to infection with ICAM-1-bearing virions, as exemplified by a more important virus replication, an increase in integrated viral DNA copies, and a more efficient entry process. Interactions between virus-associated host ICAM-1 and cell surface LFA-1 under a cluster formation seem to be responsible for the preferential HIV-1 infection of the memory cell subset. Altogether, these data shed light on a potential mechanism by which HIV-1 preferentially targets long-lived memory CD4+ T cells.     

Memory CD4(+) T cells are the earliest detectable human immunodeficiency virus type 1 (HIV-1)-infected cells in the female genital mucosal tissue during HIV-1 transmission in an organ culture system

The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4(+) T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1 gag-pol mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4(+) T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4(+) T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA [9-[2-(phosphonomethoxy)propyl] adenine], an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.