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Quantitative Analysis of Human Immunodeficiency Virus Type 1 DNA Dynamics by Real-Time PCR: Integration Efficiency in Stimulated and Unstimulated Peripheral Blood Mononuclear Cells

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Abstract

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 (ΔVpr)-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 ΔVpr-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.

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Authors and Affiliations

  1. Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japan

    Youichi Suzuki, Naoko Misawa, Hirotaka Ebina & Yoshio Koyanagi

  2. Japanese Foundation for AIDS Prevention, Tokyo, Japan

    Youichi Suzuki

  3. Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

    Chihiro Sato & Naoki Yamamoto

  4. Department of Immunotherapeutics, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

    Takao Masuda

Authors
  1. Youichi Suzuki
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  2. Naoko Misawa
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  3. Chihiro Sato
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  4. Hirotaka Ebina
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  5. Takao Masuda
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  6. Naoki Yamamoto
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  7. Yoshio Koyanagi
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Correspondence to Yoshio Koyanagi.

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Suzuki, Y., Misawa, N., Sato, C. et al. Quantitative Analysis of Human Immunodeficiency Virus Type 1 DNA Dynamics by Real-Time PCR: Integration Efficiency in Stimulated and Unstimulated Peripheral Blood Mononuclear Cells. Virus Genes 27, 177–188 (2003). https://doi.org/10.1023/A:1025732728195

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  • DOI: https://doi.org/10.1023/A:1025732728195

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