Unintegrated HIV-1 DNA Research Articles

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

Antiretroviral Therapy Is Associated with a Decrease in Unintegrated HIV-1 DNA in Pediatric Patients

Good markers for monitoring the efficacy of antiretroviral therapy in children do not currently exist. This study examined the effect of antiretroviral therapy on human immunodeficiency virus (HIV-1) unintegrated DNA (uDNA), integrated DNA (iDNA), percent uDNA, immune complex dissociated (ICD) p24 antigenemia, and plasma viral titer. Seven children were followed at therapy initiation and at approximately 3- and 10-month intervals. HIV-1 uDNA was detected in all children prior to start of therapy (average percent uDNA, 43%). At 3 months, the percent HIV uDNA decreased in all patients to an average of 18% (p = 0.01) and at 10 months decreased to an average of 1%. In contrast, the amount of HIV iDNA was relatively constant after initiation of therapy. ICD HIV p24 antigen was detected in all patients prior to therapy (average, 538 pg/ml). Over the study period, the ICD p24 antigen level decreased in three patients and remained relatively unchanged in four patients. Plasma cultures of HIV-1 were positive in only one of the seven patients prior to therapy. Among the methods evaluated, measurement of uDNA was the only parameter which reliable decreased after initiation of nucleoside therapy.

HIV-1 Reverse Transcription A Termination Step at the Center of the Genome

During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication

Quantitation of unintegrated HIV-1 DNA in asymptomatic patients in the presence or absence of antiretroviral therapy.

The objective of this work was to determine the amount of unintegrated human immunodeficiency virus (HIV) DNA (HIV uDNA) in asymptomatic individuals in the presence or absence of antiretroviral therapy. Twenty-one healthy seropositive individuals with no history of any opportunistic infection or previous use of nucleoside antiretrovirals, and 9 similarly asymptomatic individuals who had initiated nucleoside antiretroviral therapy within the last 24 months were studied. All patients had CD4 lymphocyte counts above 400/microliters. All subjects administered antiretrovirals received 400-600 mg of zidovudine daily for 2-24 months. Two individuals additionally received 400 mg of dideoxyinosine (ddI) daily for 4 and 5 months. Patient peripheral blood mononuclear cells (PBMCs) were examined for integrated and unintegrated HIV DNA by a quantitative PCR assay. In addition, CD4 counts were measured, and free and immune complex dissociated p24 antigen was detected in plasma by ELISA. The mean percentage of HIV uDNA in asymptomatic individuals not on therapy was 59%, with 95% confidence limits from 50 to 69%. In contrast, patients on therapy had a mean of only 13% HIV uDNA, with confidence limits from 2 to 25% (p < 0.001). These findings indicate that a significant amount of HIV DNA in infected, healthy patients not on therapy is in the unintegrated form, and that the amount of HIV uDNA in asymptomatic patients on nucleoside therapy is much less. The amount of HIV uDNA in PBMCs deserves further study as a new marker of the efficacy of antiretroviral therapy.

The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA

During reverse transcription the synthesis of plus strand viral DNA is initiated from an RNA polypurine primer immediately upstream of the U 3 region. The polypurine tract (PPT) sequence at this site is in HIV-1 also present in the middle of the genome. Here we demonstrate that a subpopulation of linear unintegrated HIV-1 DNA has a discontinuity in the plus strand within less than 50 bp from this central PPT, consistent with its utilization as a plus strand initiation site.

High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients

In the host cell, retroviral DNAs exist in three main forms: unintegrated linear, unintegrated circular, and integrated (the provirus). High levels of unintegrated forms of retroviral DNA often correlate with superinfection and accompanying cytopathic effects, as, for example, in the case of feline acquired immunodeficiency. In culture, HIV-1 infection also results in high levels of unintegrated viral DNA although direct correlations with cytopathicity have not been made. The low frequency of HIV-1-infected cells in patients has made it difficult to determine the structure of the viral DNA in fresh tissue samples from AIDS patients by standard methods such as Southern hybridization. The PCR technique however, which allows the detection of viral DNA at levels far below that possible by other hybridization methods is, in its conventional form, of limited use for quantitative analysis. To study the amount and form of HIV-1 DNA in primary tissue of AIDS patients we have therefore modified the PCR method. Our results indicate that each of the three species of viral DNA are detectable in blood and brain of AIDS patients, and that in autopsy samples from patients with HIV encephalitis there is a considerably higher proportion of unintegrated viral DNA.