Geethanjali Dornadula

Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy

BACKGROUND Highly active antiretroviral therapy can effectively decrease the levels of human immunodeficiency virus type 1 (HIV-1) virions in peripheral plasma and seminal fluid of infected men. Whether the genital tract of HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable virus in the peripheral plasma harbors replication-competent virus is not known. METHODS We collected peripheral-blood and semen samples from seven men with HIV-1 infections who were receiving highly active antiretroviral therapy and who had no detectable viral RNA (fewer than 50 copies per milliliter) in plasma and analyzed the samples for cell-associated proviral DNA using a quantitative polymerase-chain-reaction assay. Replication-competent viruses were evaluated by cell-coculture assays. Proviral DNA and replication-competent virus obtained from peripheral-blood and seminal cells were also analyzed by sequencing relevant viral genes. RESULTS Despite the long-term suppression of HIV-1 RNA in the plasma of the seven men, proviral DNA was detected in seminal cells in four. Replication-competent viruses were recovered from peripheral-blood cells in three men and from the seminal cells in two of these three men. The viruses recovered from the seminal cells had no genotypic mutations suggestive of resistance to antiretroviral drugs and were macrophage-tropic, a feature that is characteristic of HIV-1 strains that are capable of being sexually transmitted. CONCLUSIONS In HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable levels of viral RNA in plasma the virus may be present in seminal cells and therefore may be capable of being transmitted sexually.

Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process

ABSTRACT Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not supportvif-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.

In a Subset of Subjects on Highly Active Antiretroviral Therapy, Human Immunodeficiency Virus Type 1 RNA in Plasma Decays from 50 to <5 Copies per Milliliter, with a Half-Life of 6 Months

ABSTRACT Three of five virally suppressed human immunodeficiency virus type I (HIV-1)-infected patients treated with highly active antiretroviral therapy and followed intensively with a supersensitive reverse transcriptase PCR assay with a lower limit of quantitation of 5 copies/ml showed statistically significant viral load decays below 50 copies/ml, with half-lives of 5 to 8 months and a mean of 6 months. This range of half-lives is consistent with the estimated half-life of the latent HIV-1 reservoir in the peripheral blood. Those patients without decay of viral load in plasma may have significant cryptic HIV-1 residual replication.

Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections.

Background HIV-1-infected men on suppressive highly active antiretroviral therapy (HAART) have a reduction of viral replication in vivo, but HIV-1 RNA is still detectable by certain ultrasensitive reverse transcriptase–PCR assays in blood plasma. Replication-competent virus can also be isolated from both peripheral blood mononuclear cells (PBMC) and seminal cells of these patients. Despite HAART, on-going in vivo infection of HIV-1-seropositive patients’ PBMC was demonstrated by the detection of episomal HIV-1 moieties, known as HIV-1 two-long terminal repeat (2-LTR) DNA circles. Methods The present study analyzes whether new cellular infections occur in vivo in seminal cells of HIV-1-infected men on suppressive HAART. PBMC and seminal cells were isolated from a cohort of HIV-1-seropositive men taking suppressive HAART (< 50 copies HIV RNA/ml blood plasma). Viral growth assays were performed in vitro, as well as semi-quantitative PCR to detect HIV-1 2-LTR circular DNA in PBMC and seminal mononuclear cells. Results Viral growth in vitro was demonstrated in 16 out of 28 (57%) patients’ PBMC, and in five patients’ seminal cells (18%). Although 18 patients’ PBMC were positive for HIV-1 2-LTR DNA circles, importantly, 2-LTR circular DNA was not detected in any semen sample, even when replication-competent HIV-1 virus had been recovered from a patients seminal cells by viral co-culture assays. Conclusions The current study suggests that in HIV-1-infected men treated with suppressive HAART, new cellular infections occur in PBMC, but that new infections do not take place in seminal cells in vivo. Thus, these findings suggest that mainly latent HIV-1 occurs in seminal cells of men on suppressive HAART, which may be a compartment-specific mechanism of residual HIV-1 disease.

Partial Rescue of the Vif-Negative Phenotype of Mutant Human Immunodeficiency Virus Type 1 Strains from Nonpermissive Cells by Intravirion Reverse Transcription

ABSTRACT Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus type I (HIV-1) and is essential for viral replication. It appears that Vif functions in the virus-producing cells and affects viral assembly. Viruses with defects in the vifgene (vif−) generated from the “nonpermissive cells” are not able to complete reverse transcription. In previous studies, it was demonstrated that defects in the vif gene also affect endogenous reverse transcription (ERT) when mild detergents were utilized to permeabilize the viral envelope. In this report, we demonstrate that defects in the vif gene have much less of an effect on ERT if detergent is not used. When ERT was driven by addition of deoxyribonucleoside triphosphates (dNTPs) at high concentrations, certain levels of plus-strand viral DNA could also be achieved. Interestingly, if vif− viruses, generated from nonpermissive cells and harboring large quantities of viral DNA generated by ERT, were allowed to infect permissive cells, they could partially bypass the block at intracellular reverse transcription, through which vif− viruses without dNTP treatment could not pass. Consequently, viral infectivity can be partially rescued from the vif− phenotype. Based on our observations, we suggest that vif defects may cause the reverse transcription complex (RT complex) to become sensitive to mild detergent treatments within HIV-1 virions and become unstable in the target cells, such that the process of reverse transcription cannot be efficiently supported. Further dissection of RT complexes of vif− viruses may be key to uncovering the molecular mechanism(s) of Vif in HIV-1 pathogenesis.

Natural endogenous reverse transcription of HIV-1

Mechanisms involved with human immunodeficiency virus type I (HIV-1) sexual transmission are not fully defined. We have demonstrated that endogenous reverse transcription of lenti-viruses can occur within the intact virion. This takes place before direct infection of the target cells. In a biochemically active process, endogenous reverse transcription occurs in HIV-1 virions in specific microenvironments. In virions without non-physiological permeabilization, endogenous reverse transcription can occur and has been entitled natural endogenous reverse transcription (NERT). This molecular mechanism dramatically increases HIV-1 infection in initially-quiescent peripheral blood mononuclear cells, as well as non-proliferating cells such as macrophages. This molecular process may augment sexually transmission of HIV-1, as HIV virion particles in genital secretions are shown to have increased endogenous reverse transcripts and NERT is potently stimulated. Further studies are necessary to determine whether this molecular mechanism is critical in vivo for sexual transmission of this human lenti-viral agent.

Viral Burden and Disease Progression in HIV-1-Infected Patients With Sickle Cell Anemia

The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV‐1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV‐1 virus, will the course of the infection be altered, resulting in a prolonged symptom‐free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV‐1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV‐1‐related disease. All SS patients with a diagnosis of HIV‐1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow‐up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV‐1 viral burden. The studies included the branched‐DNA signal amplification assay, quantitative DNA‐polymerase chain reaction (PCR), quantitative reverse transcription (RT)‐initiated–PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow‐up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV‐1–infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T‐lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long‐term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long‐term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV‐1–seropositive SS patients (44%) may be asymptomatic long‐term nonprogressors. In these patients, the CD4+ T‐lymphocyte counts remained high and their viral burdens were remarkably lower than in non‐SS HIV‐1–seropositive individuals. Whereas this study does not prove an “autosplenectomy” hypothesis, it suggests that in patients with both SS and HIV‐1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV‐1 infection may be one. Am. J. Hematol. 59:199–207, 1998.

Chemokine Receptors and the Molecular Basis for Human Immunodeficiency Virus Type 1 Entry into Peripheral Hematopoietic Stem Cells and Their Progeny

There exist at least two major coreceptors for human immunodeficiency virus (HIV)-1 entry into target cells, the CXCR-4 and CCR-5 chemokine receptors for T lymphocyte-tropic and macrophage-tropic strains of HIV-1, respectively. Highly purified human CD34 cells derived from umbilical cord blood were shown not to express CD4, CXCR-4, and CCR-5 on their cell membranes, as analyzed by immunofluorescent staining and flow cytometric analyses. However, expression of these molecules was inducible when highly purified CD34 cells underwent proliferation and differentiation along myeloid cell lineages, in the presence of suitable cocktails of hematopoietic growth factors. HIV-1 infectivity studies showed that macrophage-tropic strains of HIV-1 could efficiently infect differentiated CD34 cells. T lymphocyte-tropic strains could not infect CD34 cells before or after induction of receptors and coreceptors. These data suggest that HIV-1 infection of CD34 cells and their progeny depends on membrane expression of the critical CD4 receptor, as well as certain chemokine coreceptors.