Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART.

Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART). The current experiments indicate that prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Enzyme-linked immunosorbent assay and gene array analyses indicate significant changes in the expression of proteins and messenger RNA after treatment of cells with prostratin, consistent with phagocyte activation and differentiation. Prostratin blocks HIV-1 infection relating to down-regulation of CD4 receptor expression. The array analysis indicates a similar down-regulation of the HIV-1 coreceptors, CXCR4 and CCR5, and this may also reduce viral infectivity of treated host cells. Finally, prostratin is capable of up-regulating HIV-1 expression from CD8+ T lymphocyte-depleted peripheral blood mononuclear cells of patients undergoing HAART. This novel observation suggests the agent may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1.

Human immunodeficiency virus type 1-infected persons with residual disease and virus reservoirs on suppressive highly active antiretroviral therapy can be stratified into relevant virologic and immunologic subgroups.

A significant percentage of human immunodeficiency virus type 1 (HIV-1)-infected persons treated with highly active antiretroviral therapy (HAART) will develop plasma HIV-1-specific virion RNA levels <50 copies/mL. HIV-1-infected persons receiving virally suppressive HAART were studied with a viral outgrowth assay of the patients' peripheral blood mononuclear cells (PBMC), and a quantitative polymerase chain reaction assay was used to analyze HIV-1 2-long terminal repeat (2-LTR) circular DNA in PBMC, which indicates new HIV-1 infections of cells in vivo. Viral outgrowth in vitro correlated inversely with the level of peripheral blood CD4(+) T lymphocytes. Detection and quantitation of 2-LTR circular DNA correlated strongly with viral outgrowth patterns and inversely with CD4(+) T lymphocyte counts. Relevant subgroups of HIV-1-infected subjects on suppressive HAART with residual viral disease and reservoirs can now be stratified.

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.

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 support vif-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.

Morphologic changes in human immunodeficiency virus type 1 virions secondary to intravirion reverse transcription: evidence indicating that reverse transcription may not take place within the intact viral core.

INTRODUCTION: In the past, retroviral endogenous reverse transcription (ERT) was considered an artificial process, secondary to permeabilization of the viral envelope by detergents or amphipathic peptides. However, recently we have demonstrated that ERT may occur in a variety of lentiviruses without detergent treatment and may lead to increased infectivity of lentivirions in initially quiescent T lymphocytes and nonproliferating cells, such as macrophages. As full-length reverse transcripts could be synthesized within lentiviral particles, it is worth evaluating the potential alterations in lentiviral morphology due to the stimulation of intravirion reverse transcription. METHODS: Using quantitative DNA-polymerase chain reaction (PCR) and transmission electron microscopy (TEM), we characterized critical alterations in human immunodeficiency virus type 1 (HIV-1) virions after stimulation of intravirion reverse transcription. RESULTS: Intravirion reverse transcription in HIV-1 virions was stimulated using deoxyribonucleoside triphosphates (dNTPs) and physiologic polyamines. Our studies indicated that HIV-1 virions, in which intravirion reverse transcription was stimulated, showed dissolution of the p24-shelled viral core and absence of the core-envelope linkage (CEL) region by TEM. These changes in the structure of the core correlate with the in vitro alterations in virion infectivity on primary cells. CONCLUSIONS: Stimulation of intravirion HIV-1 reverse transcription leads to morphologic changes in the viral particles that suggest changes in the compact viral core, which is consistent with active reverse transcription before infection of target cells. Further, via this unique approach, we suggest that intravirion or intracellular reverse transcription of HIV-1 is unlikely to take place within intact viral cores made up of p24-containing outer shells. As such, these results suggest a new approach to further dissect the intravirion or intracellular reverse transcription machinery of lentiviruses.

Partial rescue of the Vif-negative phenotype of mutant human immunodeficiency virus type 1 strains from nonpermissive cells by intravirion reverse transcription.

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 vif gene (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.

Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy.

CONTEXT: Despite suppressive treatment with highly active antiretroviral therapy (HAART), replication-competent virus can still be isolated from peripheral blood mononuclear cells and genital cells of many individuals receiving suppressive HAART. OBJECTIVE: To determine whether free virion RNA can be detected in the blood plasma and/or genital tract fluids from patients receiving suppressive HAART. DESIGN: Prospective cohort study conducted from November 1998 to May 1999. SETTING: Academic medical center. PATIENTS: Human immunodeficiency virus 1-infected individuals (20 men and 2 women) shown in our laboratories to have fewer than 50 copies/mL of HIV-1 RNA in peripheral blood plasma while taking suppressive HAART. MAIN OUTCOME MEASURES: Free virion RNA levels in peripheral blood plasma and genital fluids, quantified using an ultrasensitive reverse transcriptase polymerase chain reaction able to quantify cell-free virion RNA to a lower limit of 5 copies/mL and qualitatively detect viral RNA below this level. RESULTS: In all 22 patients, residual viral RNA could be detected in the peripheral blood plasma (mean level, 17 copies/mL). The presence of viral RNA suggests that ongoing viral replication is occurring, albeit at low levels, in each patient evaluated. Viral RNA levels were lower in most patients' genital fluids compared with blood plasma and in 12 patients were undetectable. CONCLUSIONS: These data suggest that low-level replication of HIV-1 in patients taking suppressive HAART may be demonstrated not only in peripheral blood mononuclear cells but also in peripheral plasma as cell-free virion RNA. Complete ablation of viral replication may require intensification of antiretroviral therapies beyond standard suppressive HAART.

HIV-1 virions produced from replicating peripheral blood lymphocytes are more infectious than those from nonproliferating macrophages due to higher levels of intravirion reverse transcripts: implications for pathogenesis and transmission.

It has been demonstrated that intravirion reverse transcription in human immunodeficiency virus type I (HIV-1) occurs in the presence of physiological substances and that the intravirion HIV-1 reverse transcripts are important for the establishment of infection in nondividing cells. In this report, we demonstrate that the infectivity of the virus produced from replicating peripheral blood lymphocytes (PBL) is significantly higher than that of virions produced by nonproliferating macrophages, upon infection of either initially quiescent PBLs or macrophages. This directly correlated with significantly higher intravirion reverse transcripts in the virions from replicating PBLs, compared to those from macrophages. Treatment of replicating PBLs and macrophages with 3'- azido-3'- deoxythymidine resulted in decreased intravirion reverse transcripts and lower infectivity of produced virions, upon infection of initially quiescent T-cells or macrophages. Because both nonproliferating macrophages and replicating lymphocytes are the major reservoirs for HIV-1 in vivo, we propose that as the result of higher levels of intravirion reverse transcripts, the HIV-1 virions produced from actively replicating cells are more infectious than those from nonproliferating cells. As such, this scenario implies at least one molecular mechanism for the intra- and interhost transmission of HIV-1.

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.

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.

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.

Natural endogenous reverse transcription of HIV type 1.

Molecular mechanisms by which human lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are sexually transmitted have yet to be fully elucidated. It is now demonstrated that endogenous reverse transcription of lentiviruses can occur within the intact virion, before infection of target cells. This is a biochemically active process and is altered by the microenvironment to which HIV-1 virions are subjected. Stimulation of endogenous reverse transcription within virion particles, without nonphysiological permeabilization, has been called natural endogenous reverse transcription (NERT). This molecular mechanism has been shown to augment HIV-1 infection in initially quiescent peripheral blood mononuclear cells (PBMCs) as well as nonproliferating macrophages. As such, this process may be important in augmenting the sexual transmission of HIV-1, as genital secretions have been shown to stimulate NERT within HIV-1 virion particles. Further studies are planned to elucidate fully this initial molecular mechanism, which may be critical in understanding the sexual transmission of HIV-1 and therefore the spread of the AIDS pandemic.

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.

Natural endogenous reverse transcription of simian immunodeficiency virus.

It has been demonstrated that human immunodeficiency virus type 1 (HIV-1) virions are biochemically active particles, within which reverse transcription can take place even in physiological microenvironments. This process has been termed "natural endogenous reverse transcription" (NERT). In this report, we demonstrate that purified virions of simian immunodeficiency virus (SIV) also contain virus-specific DNA, which resulted from partial reverse transcription. Further, viral DNA synthesis could be initiated in SIV virions in the presence of polyamines and deoxyribonucleoside triphosphates (dNTPs), at physiological concentrations. The viral infectivity upon initially quiescent cells was significantly increased, when the levels of intravirion reverse transcripts were modulated. These data suggest that NERT of SIV may play an important role for SIV pathogenesis and transmission. In contrast to HIV-1, these hypotheses may be further directly investigated by in vivo model systems.

Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase.

Integration of viral DNA into a chromosome of the infected host cell is required for efficient replication of a retroviral genome, and this reaction is mediated by the virus-encoded enzyme integrase (IN). As IN plays a pivotal role in establishing infection during the early stages of the retroviral life cycle, it is an attractive target for therapeutic intervention. However, the lack of effective antiviral drug therapy against this enzyme has led to the testing of other novel approaches towards its inhibition. In these studies, a panel of anti-human immunodeficiency virus type 1 (anti-HIV-1) IN hybridomas has been used in the construction of single-chain variable antibody fragments (SFvs). The monoclonal antibodies produced by these hybridomas, and derived SFvs, bind to different domains within IN. We now demonstrate that intracellular expression of SFvs which bind to IN catalytic and carboxy-terminal domains results in resistance to productive HIV-1 infection. This inhibition of HIV-1 replication is observed with SFvs localized in either the cytoplasmic or nuclear compartment of the cell. The expression of anti-IN SFvs in human T-lymphocytic cells and peripheral blood mononuclear cells appears to specifically neutralize IN activity prior to integration and, thus, has an effect on the integration process itself. These data support our previous studies with an anti-HIV-1 reverse transcriptase SFv and demonstrate further that intracellularly expressed SFvs can gain access to viral proteins of the HIV-1 preintegration complex. This panel of anti-HIV-1 IN SFvs also provides the tools with which to dissect the molecular mechanism(s) directly involved in integration within HIV-1-infected cells.

Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription.

Reverse transcription of HIV-1, without detergent or amphipathic peptide-induced permeability of the viral envelope, has been demonstrated to occur in the intact HIV-1 virion. In this report, we demonstrate that the amphipathic domains in the C terminus of the transmembrane glycoprotein (gp41) account for the natural permeability of the HIV-1 envelope to deoxyribonucleoside triphosphates, the substrates for DNA polymerization. In addition, nonphysiological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorporate into, and irreversibly terminate reverse transcripts. As a result, viral infectivity is potently inhibited. Since the lentiviral envelope with these newly demonstrated characteristics can serve as a delivery pathway for anti-reverse transcription agents, we propose a unique strategy to prevent HIV-1 interand, possibly, intrahost transmission.

Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell.

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.

Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells.

Endogenous reverse transcription (ERT) of retroviruses has long been considered a somewhat artificial process which only mimics reverse transcription occurring in target cells, as detergents or amphipathic peptides have classically been used to make the envelopes of retroviruses in these reaction systems permeable. Recently, several studies suggested that ERT of human immunodeficiency virus type 1 (HIV-1) might occur without detergent treatment. However, this phenomenon could be due to damage of the retroviral envelope during the process of virion purification or freezing and thawing. In this report, intravirion HIV-1 ERT, without detergent-induced permeabilization, is demonstrated to occur in the natural microenvironments of HIV-1 virions and is not caused by artificial processes. Therefore, this stage of the viral life cycle was termed natural ERT (NERT). The efficiency of NERT in HIV-1 virions was markedly augmented by several physiological substances in the extracellular milieu, such as polyamines and deoxyribonucleoside triphosphates. In addition, HIV-1 virions in seminal plasma samples harbored dramatically higher levels of full-length or nearly full-length reverse transcripts than virions isolated from peripheral blood plasma samples of HIV-1-seropositive men. When HIV-1 virions were incubated with seminal plasma samples, infectivity in initially nondividing cells was also significantly enhanced. Thus, we suggest that HIV-1 virions are actively altered by the extracellular microenvironment and that NERT may play an important role in viral infection of nondividing cells.

Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals: direct assessment of resistance to reverse transcriptase inhibitors in vivo.

Intravirion reverse transcripts have been identified in the blood plasma of human immunodeficiency virus type 1 (HIV-1)-infected individuals. In the present studies, the kinetic processes of intravirion HIV-1 reverse transcription, in the blood plasma of HIV-1-infected persons treated with nevirapine, were investigated. Nevirapine is a nonnucleoside inhibitor of reverse transcriptase (RT) which decreases the level of HIV-1 viral particles in the blood plasma of infected individuals. By analyzing HIV-1 virions at different time points prior to and after initiation of nevirapine therapy in vivo, the levels of intravirion reverse transcripts have been demonstrated to be dramatically susceptible to this anti-RT agent, out of proportion to effects on plasma virion load. The intravirion reverse transcripts were also documented to rebound to the pretreatment levels, concomitant with the development of resistant viral mutants. In addition, the infectivity of HIV-1 virions dramatically decreased after nevirapine treatment, further indicating that the effects of this anti-RT agent begin within the cell-free virions. Since the levels of intravirion reverse transcripts were altered according to the susceptibility or resistance of the HIV-1 RT enzyme to this inhibitor, these data demonstrate that the formation of intravirion reverse transcripts is a dynamic process in vivo. Moreover, because the alteration in ratios between intravirion HIV-1 reverse transcripts and viral genomic RNA directly reflects the efficiency of reverse transcription, we propose that the determination of these ratios in the blood plasma of HIV-1-positive patients may be a useful and, most importantly, a direct assay to monitor the efficacy of anti-RT agents in vivo.

Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies.

Reverse transcription of retroviral genomic RNA in a target cell is influenced by cellular factors, including the concentration of deoxyribonucleoside triphosphates (dNTPs). In addition, recent data have demonstrated that reverse transcription can be driven within human immunodeficiency virus type 1 virions, prior to infection of a cell, by increasing extracellular concentrations of dNTPs. In attempts to increase the transduction efficiency of recombinant murine leukemia virus vectors, endogenous reverse transcription was initiated within cell-free, recombinant murine leukemia virus virions in the presence of relatively high concentrations of dNTPs. As a result, the expression of transduced genes via these retroviral vectors was increased approximately 10-fold by treatment of virions with dNTPs. Combined with our previous data, these observations suggest that virion-associated DNA synthesis can occur in diverse groups of retroviruses and positively alter retroviral infectivity. As such, these manipulations may be useful for increasing the efficiency of retrovirus-mediated gene delivery.