Abstract
BackgroundViral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.ResultsAn infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.ConclusionsThis work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.
Highlights
Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo
The activity in macrophages has been attributed to Vpx binding to a ubiquitin ligase complex containing cullin4a (CUL4), damaged DNA-binding protein 1 (DDB1), and the DDB1- and CUL4-associated factor 1 (DCAF1) [18,19,20,21]
Characterization of SIVmne Vpx mutations Previous studies of HIV-2rod Vpx demonstrated a critical role for nuclear localization of two domains encompassing a tyrosine-rich sequence within residues 65-72 and a proline-rich sequence within residues 101-112 (Figure 1) [10,11]
Summary
Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. The activity in macrophages has been attributed to Vpx binding to a ubiquitin ligase complex containing cullin4a (CUL4), damaged DNA-binding protein 1 (DDB1), and the DDB1- and CUL4-associated factor 1 (DCAF1) [18,19,20,21]. The target of ubiquitination, that restricts reverse transcription by this complex in the absence of Vpx, has recently been ascribed to SAMdomain HD-domain containing protein 1 (SAMHD1), a product of an unconventional cell-intrinsic innate immune response [22,23]. SAMHD1 appears to function as a deoxynucleoside triphosphate triphosphohydrolase [24,25] It is unclear which of these distinct activities of Vpx in quiescent cells is dominant, and whether these activities are cell-type dependent
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