Abstract
BackgroundThe 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).ResultsWe 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.ConclusionOur 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.
Highlights
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
Tyrphostin inhibited degradation of tetherin by Vpu already at 25 μM whereas Cay10577 and DRB did so in a dosedependent manner, demonstrating the importance of casein kinase II (CK-II) activity for the degrading effects of Vpu on tetherin (Fig. 1E). These results show that mutation of serine 52 to alanine (S52A) is sufficient to entirely disrupt the effect of Vpu on CD4 and establish at a single cell level that an intact CK-II phosphorylation site as well as active CK-II are important for degradation of tetherin by Vpu
We speculated that Vpu with a mutated CK-II site might not be able to counteract high levels of tetherin expression found in macrophages, but may replicate efficiently in T-cells that express low levels of tetherin
Summary
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. Recent data suggest that the S52 phosphorylation site is 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). Vpu targets CD4 for degradation in the endoplasmic reticulum [2,3,4] It promotes virion release in a cell-type dependent manner by counteracting a host restriction factor that can be induced by interferon-alpha [5]. Our data suggest that the ability of Vpu to counteract tetherin is an important determinant for HIV-1 cell tropism
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