Figure 2.

Vpr is the viral determinant responsible for PHF13 reduction. (a) SupT1 cells were infected with equal p24 amounts (200 ng) of VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP or variants with inactivating mutations in Vpr, Nef or Vpu. Forty-eight hours post-infection cell lysates were subjected to immunoblot against PHF13, HIV-1 p24, tubulin and Vpr. (b) 293T cells were either transfected with equal DNA amounts of the indicated HIV-1 NL4-3 IRES-eGFP constructs or infected with same p24 amounts (100 ng) of the respective VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP virus stocks. Thirty-six hours later cells were analysed by immunoblot for PHF13, HIV-1 p24 and tubulin levels. (c) SupT1 cells were infected with 200 ng p24 of VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP or the ΔVpr variant, transcomplemented in the 293T producer cells with Vpr or GFP only. Forty-eight hours later, cells were lysed and subjected to immunoblot for detection of PHF13, HIV-1 p24, tubulin and Vpr. In addition to p24 quantification all transfections or infections presented in (a)–(c) were analysed by flow cytometry for the % of GFP+ cells. These were similar within experiments and in the range of 50 to 90%. All immunoblots presented in (a)–(c) were confirmed in at least two additional independent experiments. (d) Primary CD4⁺ T cells from two different donors were infected with 400 ng p24 VSVG pseudotyped HIV-1 NL4-3, a variant with inactivated Vpr ORF or mock infected. Cells were harvested 48 hpi, total cell extracts were prepared and analysed for expression of PHF13, HIV-1 capsid p24, actin and Vpr by immunoblot.