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Viruses interact with multiple host cell factors. Some of these are required to promote viral propagation, others have roles in inhibiting infection. Here, we delineate the function of the cellular factor PHF13 (or SPOC1), a putative HIV-1 restriction factor. Early in the HIV-1 replication cycle PHF13 increased the number of integrated proviral copies and the number of infected cells. However, after HIV-1 integration, high levels of PHF13 suppressed viral gene expression. The antiviral activity of PHF13 is counteracted by the viral accessory protein Vpr, which mediates PHF13 degradation. Altogether, the transcriptional master regulator and chromatin binding protein PHF13 does not have purely repressive effects on HIV-1 replication, but also promotes viral integration. By the functional characterization of the dual role of PHF13 during the HIV-1 replication cycle, we reveal a surprising and intricate mechanism through which HIV-1 might regulate the switch from integration to viral gene expression. Furthermore, we identify PHF13 as a cellular target specifically degraded by HIV-1 Vpr. ispartof: Open Biology vol:7 issue:10 ispartof: location:England status: published

U2OS cells were treated with 1 μg/ml doxycycline or left untreated for 24 h before cells were infected with 100 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRESeGFP. 24 hpi cells were analyzed by flow cytometry. The mean percentage of GFP+ cells from three independent experiments was calculated and the resulting data was normalized to untreated cells (100 %).

Jurkat-TAg or SupT1 cells were infected with 200 ng p24 VSVG pseudotyped HIV-1 NL4-3 or mock infected. Cells were harvested 48 hpi, total cell extracts were prepared and analyzed for expression of PHF13, HIV-1 capsid p24 and tubulin by immunoblot.

U2OS cells were infected with 100 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP for 24 h. Thereafter, cells were treated with 1 μg/ml doxycycline.Additional 24 h later cells were analyzed by flow cytometry for the % and MFI of GFP+ (HIV-1-infected) cells. The graph shows mean values and standard deviations of three independent experiments.

U2OS-C5 cells were mock transfected or with a control siRNA (csiRNA) or directed against PHF13 (siPHF13). 24 h later cells were infected with 100 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP or the ΔVpr variant for 6 h, washed and incubated for additional 18 h. At that time point, cells were induced with 1 μg/ml doxycycline. 24 h later cells were lysed to detect PHF13, tubulin, HIV-1 p24 and Vpr by immunoblot.

The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.