Quantitative analysis of human immunodeficiency virus type 1 DNA dynamics by real-time PCR: integration efficiency in stimulated and unstimulated peripheral blood mononuclear cells.
We established a set of real-time PCR assay to accurately quantify human immunodeficiency virus type 1 (HIV-1) DNA in infected cells. Using this assay we were able to measure the strong-stop, full-length/ 1-LTR circle, 2-LTR circle, and integrated forms of viral DNA, and the data provided was quite consistent with the characteristics of mutant viruses in early phase of infection. Since our assay is particularly applicable to quantify the integrated DNA in small scale of samples, we measured the level of integrated DNA in wild-type virus (WT)- or Vpr-defective virus (deltaVpr)-infected peripheral blood mononuclear cells (PBMC), and examined whether quiescent condition of the PBMC influences integration step of HIV-1. Under stimulating condition approximately 25% of total viral DNA was in integrated form in either WT- or DeltaVpr-infected cells. In contrast, under unstimulated condition the level of integration efficiency was not significantly reduced in WT-infected cells, while this efficiency was severely impaired in the absence of vpr gene. This result clearly demonstrated a crucial role of the Vpr for nuclear localization and subsequent integration of viral DNA in nondividing cells. Therefore, our assay is useful for analyzing the events in early phase of HIV-1 infection under various conditions.
D018929:Cell Culture Techniques; D002455:Cell Division; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005814:Genes, Viral; D016334:Genes, vpr; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000502:physiology / Q000821:virology*; D020125:Mutation, Missense; D010835:Phytohemagglutinins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D017384:Sequence Deletion; D016662:Virus Integration / Q000502:physiology*
D004279:DNA, Viral; D010835:Phytohemagglutinins; C041124:strong-stop DNA