Vpu serine 52 dependent counteraction of tetherin is required for HIV-1 replication in macrophages, but not in ex vivo human lymphoid tissue

Abstract

Background: The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).

Results: We show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.

Conclusion: Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.

Figure 1

Mutation of S52A impairs Vpu-mediated degradation of CD4 and tetherin. (A) Western blot analysis of Vpu expression in lysates of transfected 293T cells. (B) FACS analysis of CD4 expression by Jurkat (upper panel) and CD4 co-transfected 293T cells (lower panel) expressing GFP alone or together with the Vpu and Vpu S52A proteins. Numbers give the MFI of the specified region (C) Quantitative analysis of CD4 downmodulation in Jurkat and 293T cells. Shown are the mean percentages of CD4 down-modulation +/- SD from six (Jurkat) and three (293T) independent experiments. Cell surface CD4 is given as a percentage of that measured on cells transfected with the control vector expressing GFP only (100%). (D) Quantitative analysis of tetherin degradation in 293T cells. Numbers give percentages of GFP+/eCFP+ cells in the specified region. Shown are the mean percentages of tetherin degradation from eight independent transfections. Values give percentages of cells co-expressing GFP and eCFP-tetherin. The mean values obtained with the GFP only control are set as 100%. (E) The same experimental setup as presented in D, however with different concentrations of the indicated CK-II inhibitors added during media change following transfection. Means and standard deviations are calculated from three to six independent transfection experiments.

Figure 2

Vpu S52A dose-dependently counteracts tetherin in transfected 293T cells. (A) WB analysis of cellular lysates transfected with the indicated HIV-1 proviral constructs and different concentrations of tetherin plasmid. Viral supernatants were harvested two days post transfection, filtered and pelleted. Lysed cells and virus stocks were blotted for the presence of p24 and actin as a loading control. (B) Quantification of p24 release by the proviral constructs in the presence of different amounts of tetherin and analysis of tetherin transfected 293T cells. Presented is one out of two independent WB experiments showing the same results. Abbreviations, U-, Vpu-defective; S52A, VpuS52A. (C) Western blot analysis of endogenous tetherin expression in PBL and MDM from three different donors. PBL were either left untreated or stimulated with 1 μg/ml PHA for 24 hours (PBL+).

Figure 3

Vpu S52A does not impair HIV-1 release from P4-CCR5 cells. (A) Western blot analysis of viral gene expression in lysates of transfected 293T cells. (B) Viral particle release by P4-CCR5 cells transfected with the indicated X4 and R5 HIV-1 NL4-3 proviral constructs. P4-CCR5 cells were transfected with 0.1 μg proviral DNA in sextuplicates and p24 in the culture supernatants was quantified by p24 ELISA three days later. Measurement of the β-Gal activities in the cell lysates verified similar transfection efficiencies (not shown). Values give averages +/- SD from two independent experiments with sextuplicate transfections and represent percentages compared to NL4-3 wildtype transfected cells (100%). (C) P4-CCR5 indicator cells were infected in triplicate with virus stocks containing 1 ng p24 antigen derived from 293T cells transfected with the indicated proviral constructs and β-Gal activity was determined three days later. Shown are average values +/- SD from two independent experiments with triplicate infections of two independent virus stocks. Infectivity is given as percentage compared to infectivity of NL4-3 wildtype infected cells (100%). Abbreviations, N-, Nef-defective; U-, Vpu-defective; S52A, VpuS52A.

Figure 4

Vpu S52A is dispensable for HIV-1 replication and cytopathicity in ex vivo infected HLT. Representative replication kinetics (A) of the indicated X4 and R5 HIV-1 NL4-3 constructs. (B) Cumulative p24 production over 15 days and (C) CD4+ T cells depletion at the end of culture in tissues from eight (X4) and ten (R5) donors infected with the indicated HIV-1 variants. Values are given as percentages compared to cultures infected with NL4-3 wildtype (100%). Shown are means +/- SEM. (D) Correlation between p24 production and CD4+ T-cell depletion.

Figure 5

Vpu S52A impairs HIV-1 replication in macrophages. Replication kinetics of wildtype NL4-3 and the indicated mutants in monocyte-derived macrophages and average levels of cumulative RT production by macrophages infected with the NL4-3 variants over a 20 day period. Values give averages +/- SEM of macrophages from three different donors with two independent virus stocks containing 1 ng p24 antigen. PSL, photon-stimulated luminescence.

Figure 6

Modulation of tetherin and CD4 in primary T-cells and macrophages by Vpu. (A) FACS analysis of CD4 and tetherin modulation in infected PBL cultures. PBL were infected with HIV-1 variants expressing eGFP via an IRES. Cells were stained with antibodies and measured by flow cytometry three days later. To quantify modulation of cell surface expressed CD4 and tetherin MFI of PBLs infected with HIV-1 NL4-3 WT was set as 100%. Depicted are means +/- SD derived from experiments with four different donors. (B) Primary macrophages were infected with the indicated R5-tropic virus stocks expressing eGFP via an IRES. Cells were analyzed for cell surface MHC-I, CD4 and tetherin five days post infection similar to the PBL cultures. Presented are means +/- SD from infections with macrophages from three different donors each of those were infected with two independent virus stocks.