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A CRISPR/Cas9 approach reveals that the polymerase activity of DNA Polymerase β is dispensable for HIV-1 infection in dividing and nondividing cells.

A CRISPR/Cas9 approach reveals that the polymerase activity of DNA Polymerase β is dispensable for HIV-1 infection in dividing and nondividing cells.
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Goetze RW, Kim DH, Schinazi RF, Kim B,


Goetze RW, Kim DH, Schinazi RF, Kim B, (click to view)

Goetze RW, Kim DH, Schinazi RF, Kim B,

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The Journal of biological chemistry 2017 07 06() pii 10.1074/jbc.M117.793661

Abstract

Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase β (Pol β). However, whether human Pol β; is essential for lentivirus replication in human cells is unclear. Here, we abolished Pol β expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate Pol β’s role in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol β-knockout was confirmed by enhanced sensitivity to methyl methanesulfonate – induced DNA damage. Of note, nuclear extracts from Pol β-knockout THP-1 cells prepared from both dividing and nondividing stages displayed significantly reduced capability to repair the gapped HIV-1 integration intermediate DNA substrate in a biochemical simulation. However, nuclear extract from both dividing and nondividing stages of the Pol β-KO cells had detectable gap repair activity, suggesting that other host DNA polymerases also repair gapped HIV-1 DNA, particularly in dividing cells. Next, when we compared transduction using HIV-1 and simian immunodeficiency virus in control and Pol β-KO cells, the loss of the Pol β expression did not affect transduction efficiency of these lentiviruses in both dividing and nondividing stages. Finally, the gap repair assay indicated that limited cellular dNTP pools, but not Pol β expression, are a primary factor for HIV-1 DNA gap repair, particularly in nondividing cells. These data support the idea that Pol β polymerase activity is dispensable for HIV-1 infection in both dividing and nondividing stages of human cells targeted by the virus.

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