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The Expression of Functional Vpx during Pathogenic SIVmac Infections of Rhesus Macaques Suppresses SAMHD1 in CD4+ Memory T Cells.

Shingai M, Welbourn S, Brenchley JM, Acharya P, Miyagi E, Plishka RJ, Buckler-White A, Kwong PD, Nishimura Y, Strebel K, Martin MA - PLoS Pathog. (2015)

Bottom Line: Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage.Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated.These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.

No MeSH data available.


Related in: MedlinePlus

The presence of wild type Vpx correlates with reduced levels of endogenous SAMHD1 in ConA-activated macaque CD4+ T lymphocytes.CD8+ T cell-depleted rhesus PBMCs were activated with ConA for 24 h, cultured with IL-2 containing medium for 48 h, negatively selected for CD4+ T cells, and infected with SIVmac239 (MOI = 0.2). Cells and supernatant media were collected daily from the infected or mock-infected “enriched” CD4+ T lymphocyte cultures. (A) Progeny virions released into the medium were detected by 32P RT assay. (B) Levels of endogenous SAMHD1 present in SIVmac239 or mock infected cell extracts from 3 x 105 cells were determined by immunoblotting following polyacrylamide gel electrophoresis using anti-SAMHD1 antibody. GAPDH was used as a loading control. (C) Individual CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or corresponding Vpx derived mutants (MOI = 0.2). Based on the results shown in Panels A and B, cells were harvested on day 3 PI and levels of endogenous SAMHD1 present in virus or mock infected cell extracts were determined by immunoblotting. (D) SAMHD1 phosphorylation status in macaque CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with/without Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (from 3 × 105 cells) of non-activated and ConA-activated CD4+ T lymphocytes from two uninfected animals were separated on acrylamide gels with/without Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 and GAPDH antibodies.
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ppat.1004928.g003: The presence of wild type Vpx correlates with reduced levels of endogenous SAMHD1 in ConA-activated macaque CD4+ T lymphocytes.CD8+ T cell-depleted rhesus PBMCs were activated with ConA for 24 h, cultured with IL-2 containing medium for 48 h, negatively selected for CD4+ T cells, and infected with SIVmac239 (MOI = 0.2). Cells and supernatant media were collected daily from the infected or mock-infected “enriched” CD4+ T lymphocyte cultures. (A) Progeny virions released into the medium were detected by 32P RT assay. (B) Levels of endogenous SAMHD1 present in SIVmac239 or mock infected cell extracts from 3 x 105 cells were determined by immunoblotting following polyacrylamide gel electrophoresis using anti-SAMHD1 antibody. GAPDH was used as a loading control. (C) Individual CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or corresponding Vpx derived mutants (MOI = 0.2). Based on the results shown in Panels A and B, cells were harvested on day 3 PI and levels of endogenous SAMHD1 present in virus or mock infected cell extracts were determined by immunoblotting. (D) SAMHD1 phosphorylation status in macaque CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with/without Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (from 3 × 105 cells) of non-activated and ConA-activated CD4+ T lymphocytes from two uninfected animals were separated on acrylamide gels with/without Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 and GAPDH antibodies.

Mentions: As noted earlier, a majority of previous studies evaluating virion-associated Vpx-mediated degradation of SAMHD1 have been conducted in non-lymphoid cells using pseudotyped virus preparations capable of only single cycles of replication. To determine whether Vpx-mediated degradation of endogenous SAMHD1 occurred during the course of spreading infections of replication-competent virus in CD4+ T lymphocytes, freshly collected, and negatively selected, Con-A stimulated rhesus macaque CD4+ T cells were infected with WT SIVmac239 at a multiplicity of infection (MOI) = 0.2. Cells and supernatant samples were collected daily and examined for levels of progeny virions released into the medium (32P-reverse transcriptase [RT] activity) and endogenous SAMHD1 (immunoblotting). Newly produced virus first became detectable on day 2 post infection (PI) and steadily increased on days 3 and 4 (Fig 3A). The levels of endogenous SAMHD1, present in the CD4+ T cells on days 1 and 2, markedly declined on days 3 and 4 PI when compared to an unrelated cellular protein (GAPDH) (Fig 3B). In an independent experiment, also assessing the status of endogenous SAMHD1, ConA-activated rhesus CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or the two different and corresponding Vpx defective mutants, all at a MOI = 0.2. Based on the results of the experiment shown in Fig 3A and 3B, cells were collected on day 3 PI and lysates were examined by immunoblotting for levels of endogenous SAMHD1. As shown in Fig 3C, Con A-stimulated rhesus CD4+ T cells, infected with both of the WT SIVmac viruses, contained reduced levels of endogenous SAMHD1 compared to levels in mock infected cells or in cells infected with the corresponding Vpx mutant viruses. Together, these results demonstrate that SIV Vpx mutants bearing the Q76A point mutation, which specifically blocks the recruitment of DCAF1, are defective in degrading endogenous SAMHD1 (Fig 3) and are attenuated during spreading infections in cultured activated PBMC (Fig 2) compared to their WT counterparts.


The Expression of Functional Vpx during Pathogenic SIVmac Infections of Rhesus Macaques Suppresses SAMHD1 in CD4+ Memory T Cells.

Shingai M, Welbourn S, Brenchley JM, Acharya P, Miyagi E, Plishka RJ, Buckler-White A, Kwong PD, Nishimura Y, Strebel K, Martin MA - PLoS Pathog. (2015)

The presence of wild type Vpx correlates with reduced levels of endogenous SAMHD1 in ConA-activated macaque CD4+ T lymphocytes.CD8+ T cell-depleted rhesus PBMCs were activated with ConA for 24 h, cultured with IL-2 containing medium for 48 h, negatively selected for CD4+ T cells, and infected with SIVmac239 (MOI = 0.2). Cells and supernatant media were collected daily from the infected or mock-infected “enriched” CD4+ T lymphocyte cultures. (A) Progeny virions released into the medium were detected by 32P RT assay. (B) Levels of endogenous SAMHD1 present in SIVmac239 or mock infected cell extracts from 3 x 105 cells were determined by immunoblotting following polyacrylamide gel electrophoresis using anti-SAMHD1 antibody. GAPDH was used as a loading control. (C) Individual CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or corresponding Vpx derived mutants (MOI = 0.2). Based on the results shown in Panels A and B, cells were harvested on day 3 PI and levels of endogenous SAMHD1 present in virus or mock infected cell extracts were determined by immunoblotting. (D) SAMHD1 phosphorylation status in macaque CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with/without Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (from 3 × 105 cells) of non-activated and ConA-activated CD4+ T lymphocytes from two uninfected animals were separated on acrylamide gels with/without Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 and GAPDH antibodies.
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ppat.1004928.g003: The presence of wild type Vpx correlates with reduced levels of endogenous SAMHD1 in ConA-activated macaque CD4+ T lymphocytes.CD8+ T cell-depleted rhesus PBMCs were activated with ConA for 24 h, cultured with IL-2 containing medium for 48 h, negatively selected for CD4+ T cells, and infected with SIVmac239 (MOI = 0.2). Cells and supernatant media were collected daily from the infected or mock-infected “enriched” CD4+ T lymphocyte cultures. (A) Progeny virions released into the medium were detected by 32P RT assay. (B) Levels of endogenous SAMHD1 present in SIVmac239 or mock infected cell extracts from 3 x 105 cells were determined by immunoblotting following polyacrylamide gel electrophoresis using anti-SAMHD1 antibody. GAPDH was used as a loading control. (C) Individual CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or corresponding Vpx derived mutants (MOI = 0.2). Based on the results shown in Panels A and B, cells were harvested on day 3 PI and levels of endogenous SAMHD1 present in virus or mock infected cell extracts were determined by immunoblotting. (D) SAMHD1 phosphorylation status in macaque CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with/without Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (from 3 × 105 cells) of non-activated and ConA-activated CD4+ T lymphocytes from two uninfected animals were separated on acrylamide gels with/without Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 and GAPDH antibodies.
Mentions: As noted earlier, a majority of previous studies evaluating virion-associated Vpx-mediated degradation of SAMHD1 have been conducted in non-lymphoid cells using pseudotyped virus preparations capable of only single cycles of replication. To determine whether Vpx-mediated degradation of endogenous SAMHD1 occurred during the course of spreading infections of replication-competent virus in CD4+ T lymphocytes, freshly collected, and negatively selected, Con-A stimulated rhesus macaque CD4+ T cells were infected with WT SIVmac239 at a multiplicity of infection (MOI) = 0.2. Cells and supernatant samples were collected daily and examined for levels of progeny virions released into the medium (32P-reverse transcriptase [RT] activity) and endogenous SAMHD1 (immunoblotting). Newly produced virus first became detectable on day 2 post infection (PI) and steadily increased on days 3 and 4 (Fig 3A). The levels of endogenous SAMHD1, present in the CD4+ T cells on days 1 and 2, markedly declined on days 3 and 4 PI when compared to an unrelated cellular protein (GAPDH) (Fig 3B). In an independent experiment, also assessing the status of endogenous SAMHD1, ConA-activated rhesus CD4+ T lymphocyte cultures were infected with WT SIVmac239, WT SIVmac316, or the two different and corresponding Vpx defective mutants, all at a MOI = 0.2. Based on the results of the experiment shown in Fig 3A and 3B, cells were collected on day 3 PI and lysates were examined by immunoblotting for levels of endogenous SAMHD1. As shown in Fig 3C, Con A-stimulated rhesus CD4+ T cells, infected with both of the WT SIVmac viruses, contained reduced levels of endogenous SAMHD1 compared to levels in mock infected cells or in cells infected with the corresponding Vpx mutant viruses. Together, these results demonstrate that SIV Vpx mutants bearing the Q76A point mutation, which specifically blocks the recruitment of DCAF1, are defective in degrading endogenous SAMHD1 (Fig 3) and are attenuated during spreading infections in cultured activated PBMC (Fig 2) compared to their WT counterparts.

Bottom Line: Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage.Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated.These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.

No MeSH data available.


Related in: MedlinePlus