ABSTRACT
BACKGROUND: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. OBJECTIVE: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. STUDY DESIGN: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. RESULTS/CONCLUSIONS: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.
Subject(s)
DNA, Viral/isolation & purification , HIV Infections/virology , HIV-1/isolation & purification , Leukocytes, Mononuclear/virology , Organic Chemicals , Polymerase Chain Reaction/methods , Proviruses/isolation & purification , Benzothiazoles , Blood Donors , CD4 Lymphocyte Count , DNA, Viral/analysis , Diamines , HIV Seropositivity/virology , HIV-1/genetics , Humans , Quinolines , RNA, Viral/blood , Sensitivity and Specificity , Viral Load , ViremiaABSTRACT
HIV-1 viral load represents a basic marker for evaluation of the rate and severity of HIV-1 related disease and to monitor the effectiveness of treatment. An SYBR green-based real-time RT-PCR (SYBR green real-time RT-PCR) revealed by Light Cycler technology was evaluated for quantitation of HIV-1 RNA viral load in plasma of HIV-1 seropositive patients. The performance of the SYBR green real-time PCR was assessed on 56 HIV-1 seropositive patients under highly active retroviral therapy (HAART) and 25 blood donors. The results demonstrated that this technique detected 50 HIV-1 RNA copies per millilitre of plasma. Moreover, we compared real-time RT-PCR with the b-DNA technique considered widely a reference technique for HIV-1 RNA viral load measurement. The parallel quantitative analysis of HIV-1 positive samples showed a high correlation (r=0.908) between the two methods. Although b-DNA and the real-time-based method gave similar sensitivity, the assay determined quantitatively HIV-1 RNA copies in 4 out of 16 samples shown as undetectable by b-DNA. The SYBR green real-time RT-PCR represents a good alternative to b-DNA assay in HIV-1 viral load determination especially during the monitoring of HAART treatment.
Subject(s)
HIV-1/genetics , HIV-1/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Virology/methods , Antiretroviral Therapy, Highly Active , Base Sequence , CD4 Lymphocyte Count , DNA Primers/genetics , Fluorescent Dyes , HIV Seronegativity , HIV Seropositivity/drug therapy , HIV Seropositivity/immunology , HIV Seropositivity/virology , Humans , RNA, Viral/blood , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Viremia/virology , Virology/statistics & numerical dataABSTRACT
Several studies have indicated that human immunodeficiency virus type-1 (HIV-1) transactivating Tat protein is essential for proviral DNA transcription and virus replication. In addition, it is actively released from acutely HIV-1-infected cells and interacts either with the same virus-infected and virus producing cell, or with bystander uninfected cells, influencing the expression of several genes and related cellular functions. The main goal of this paper was to determine the Tat-related expression of basic cellular genes in a permanently tat transfected CD4+ cell line, to identify the cellular genes influenced by the presence of endogenous-exogenous Tat protein. For this purpose, we analyzed, by a cDNA-membrane-array assay, cellular mRNAs expressed in serum-free cultures of lymphoblastoid CD4(+) Jurkat cells, stably transfected with a plasmid constitutively expressing tat gene, in comparison with Jurkat cells transfected with the backbone plasmid only, and parental Jurkat cells. The expression of mRNAs in permanently tat-transfected Jurkat cells showed significant differences in 24 out of 1176 analyzed genes in comparison with parental or backbone plasmid transfected cells. Most of the genes overexpressed in permanently tat-transfected Jurkat cells, belong to transcription factors, or to receptors, adaptors, and mediators of signal transduction pathways, and to factors involved in response to oxidative stress, suggesting a complex regulation of CD4(+) T-lymphoid cell survival and proliferation by HIV-1 Tat protein.
