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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

WT SIVmac239 degrades endogenous SAMHD1 in memory CD4+ T cells during the acute infection of rhesus macaques.(A) Levels of endogenous SAMHD1 expression were examined by immunoblotting rhesus macaque naïve and memory CD4+ T cells in blood and spleen, and myeloid lineages (CD14+ cells, MDM, and alveolar macrophages) or in two control human T cell leukemia lines (PM1 and SupT1-R5). Whole-cell extracts (from 3 × 105 cells/lane) from each indicated source were separated on 10% acrylamide gels and stained using anti-SAMHD1 antibody. GAPDH was used as a loading control. The numbers below each lane indicate relative densitometric intensities of SAMHD1 bands relative to that of GAPDH and normalized to that present in PM1 cells. (B) SAMHD1 phosphorylation status in macaque memory CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (3 × 105 cells) from PM1 cells, freshly collected rhesus CD4+ T cells or sorted rhesus memory CD4+ T cells from blood or spleen were separated on acrylamide gels with Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 antibody. (C) Levels of SAMHD1 in sorted rhesus memory CD4+ T cells infected with VSV-G pseudotyped WT SIVmac239, SIVmac239 X-del or SIVmac239 X-Q76A were assessed by immunoblotting using anti-SAMHD1 antibody. (D) Acute phase viremia in two macaques inoculated intravenously with 10,000 TCID50 of SIVmac239. (E) Memory CD4+ T cells were prepared by cell sorting from PBMC or spleen from an uninfected or an infected rhesus macaque at day 9 PI following IV inoculation with 10,000 TCID50 of SIVmac239. The plasma viral load on day 9 post infection in the macaque inoculated with WT SIVmac239 was 7.8 x 107 RNA copies/ml; viral RNA in the uninfected animals were below levels of detection (< 100 RNA copies/ml). Whole-cell extracts from 3 x 105 sorted memory CD4+ T cells from each source or from PM1 cells were analyzed by immunoblotting using anti-SAMHD1 and anti-GAPDH antibodies.
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ppat.1004928.g004: WT SIVmac239 degrades endogenous SAMHD1 in memory CD4+ T cells during the acute infection of rhesus macaques.(A) Levels of endogenous SAMHD1 expression were examined by immunoblotting rhesus macaque naïve and memory CD4+ T cells in blood and spleen, and myeloid lineages (CD14+ cells, MDM, and alveolar macrophages) or in two control human T cell leukemia lines (PM1 and SupT1-R5). Whole-cell extracts (from 3 × 105 cells/lane) from each indicated source were separated on 10% acrylamide gels and stained using anti-SAMHD1 antibody. GAPDH was used as a loading control. The numbers below each lane indicate relative densitometric intensities of SAMHD1 bands relative to that of GAPDH and normalized to that present in PM1 cells. (B) SAMHD1 phosphorylation status in macaque memory CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (3 × 105 cells) from PM1 cells, freshly collected rhesus CD4+ T cells or sorted rhesus memory CD4+ T cells from blood or spleen were separated on acrylamide gels with Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 antibody. (C) Levels of SAMHD1 in sorted rhesus memory CD4+ T cells infected with VSV-G pseudotyped WT SIVmac239, SIVmac239 X-del or SIVmac239 X-Q76A were assessed by immunoblotting using anti-SAMHD1 antibody. (D) Acute phase viremia in two macaques inoculated intravenously with 10,000 TCID50 of SIVmac239. (E) Memory CD4+ T cells were prepared by cell sorting from PBMC or spleen from an uninfected or an infected rhesus macaque at day 9 PI following IV inoculation with 10,000 TCID50 of SIVmac239. The plasma viral load on day 9 post infection in the macaque inoculated with WT SIVmac239 was 7.8 x 107 RNA copies/ml; viral RNA in the uninfected animals were below levels of detection (< 100 RNA copies/ml). Whole-cell extracts from 3 x 105 sorted memory CD4+ T cells from each source or from PM1 cells were analyzed by immunoblotting using anti-SAMHD1 and anti-GAPDH antibodies.

Mentions: In vivo, the principal target of both HIV-1 and SIVmac are memory CD4+ T lymphocytes [11,22,23]. The levels of SAMHD1 in unstimulated rhesus macaque memory CD4+ T cells, freshly collected from either blood or spleen, were examined by immunoblotting using anti-human SAMHD1 antibody. As shown in Fig 4A, higher levels of SAMHD1 were detected in memory CD4+ T lymphocytes than those measured in naïve CD4+ T cells from the same two sources. Among rhesus myeloid lineages, circulating CD14+ monocytes expressed levels of SAMHD1 similar to those present in memory CD4+ T cells, whereas much higher concentrations of SAMHD1 were detected in macaque alveolar macrophage, collected by bronchoalveolar lavage, and rhesus MDM derived in vitro. Cell lysates from the same preparations of freshly collected unstimulated rhesus macaque memory CD4+ T cells from blood or spleen were subjected to electrophoresis in Phos-tag Acrylamide and immunoblotting using the anti-SAMHD1 antibody. As shown in Fig 4B, most of the SAMHD1 in memory CD4+ T cells was unphosphorylated. The relatively high levels of expression of unphosphorylated endogenous SAMHD1 measured in memory CD4+ T lymphocytes suggested that SAMHD1 might significantly restrict viral infections in vivo, reduce virus production systemically, and affect disease progression if not adequately suppressed by Vpx.


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)

WT SIVmac239 degrades endogenous SAMHD1 in memory CD4+ T cells during the acute infection of rhesus macaques.(A) Levels of endogenous SAMHD1 expression were examined by immunoblotting rhesus macaque naïve and memory CD4+ T cells in blood and spleen, and myeloid lineages (CD14+ cells, MDM, and alveolar macrophages) or in two control human T cell leukemia lines (PM1 and SupT1-R5). Whole-cell extracts (from 3 × 105 cells/lane) from each indicated source were separated on 10% acrylamide gels and stained using anti-SAMHD1 antibody. GAPDH was used as a loading control. The numbers below each lane indicate relative densitometric intensities of SAMHD1 bands relative to that of GAPDH and normalized to that present in PM1 cells. (B) SAMHD1 phosphorylation status in macaque memory CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (3 × 105 cells) from PM1 cells, freshly collected rhesus CD4+ T cells or sorted rhesus memory CD4+ T cells from blood or spleen were separated on acrylamide gels with Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 antibody. (C) Levels of SAMHD1 in sorted rhesus memory CD4+ T cells infected with VSV-G pseudotyped WT SIVmac239, SIVmac239 X-del or SIVmac239 X-Q76A were assessed by immunoblotting using anti-SAMHD1 antibody. (D) Acute phase viremia in two macaques inoculated intravenously with 10,000 TCID50 of SIVmac239. (E) Memory CD4+ T cells were prepared by cell sorting from PBMC or spleen from an uninfected or an infected rhesus macaque at day 9 PI following IV inoculation with 10,000 TCID50 of SIVmac239. The plasma viral load on day 9 post infection in the macaque inoculated with WT SIVmac239 was 7.8 x 107 RNA copies/ml; viral RNA in the uninfected animals were below levels of detection (< 100 RNA copies/ml). Whole-cell extracts from 3 x 105 sorted memory CD4+ T cells from each source or from PM1 cells were analyzed by immunoblotting using anti-SAMHD1 and anti-GAPDH antibodies.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440783&req=5

ppat.1004928.g004: WT SIVmac239 degrades endogenous SAMHD1 in memory CD4+ T cells during the acute infection of rhesus macaques.(A) Levels of endogenous SAMHD1 expression were examined by immunoblotting rhesus macaque naïve and memory CD4+ T cells in blood and spleen, and myeloid lineages (CD14+ cells, MDM, and alveolar macrophages) or in two control human T cell leukemia lines (PM1 and SupT1-R5). Whole-cell extracts (from 3 × 105 cells/lane) from each indicated source were separated on 10% acrylamide gels and stained using anti-SAMHD1 antibody. GAPDH was used as a loading control. The numbers below each lane indicate relative densitometric intensities of SAMHD1 bands relative to that of GAPDH and normalized to that present in PM1 cells. (B) SAMHD1 phosphorylation status in macaque memory CD4+ T lymphocytes was determined by electrophoresis in acrylamide gels with Phos-Tag and analyzed by immunoblotting. Whole-cell extracts (3 × 105 cells) from PM1 cells, freshly collected rhesus CD4+ T cells or sorted rhesus memory CD4+ T cells from blood or spleen were separated on acrylamide gels with Phos-Tag and analyzed by immunoblotting using anti-SAMHD1 antibody. (C) Levels of SAMHD1 in sorted rhesus memory CD4+ T cells infected with VSV-G pseudotyped WT SIVmac239, SIVmac239 X-del or SIVmac239 X-Q76A were assessed by immunoblotting using anti-SAMHD1 antibody. (D) Acute phase viremia in two macaques inoculated intravenously with 10,000 TCID50 of SIVmac239. (E) Memory CD4+ T cells were prepared by cell sorting from PBMC or spleen from an uninfected or an infected rhesus macaque at day 9 PI following IV inoculation with 10,000 TCID50 of SIVmac239. The plasma viral load on day 9 post infection in the macaque inoculated with WT SIVmac239 was 7.8 x 107 RNA copies/ml; viral RNA in the uninfected animals were below levels of detection (< 100 RNA copies/ml). Whole-cell extracts from 3 x 105 sorted memory CD4+ T cells from each source or from PM1 cells were analyzed by immunoblotting using anti-SAMHD1 and anti-GAPDH antibodies.
Mentions: In vivo, the principal target of both HIV-1 and SIVmac are memory CD4+ T lymphocytes [11,22,23]. The levels of SAMHD1 in unstimulated rhesus macaque memory CD4+ T cells, freshly collected from either blood or spleen, were examined by immunoblotting using anti-human SAMHD1 antibody. As shown in Fig 4A, higher levels of SAMHD1 were detected in memory CD4+ T lymphocytes than those measured in naïve CD4+ T cells from the same two sources. Among rhesus myeloid lineages, circulating CD14+ monocytes expressed levels of SAMHD1 similar to those present in memory CD4+ T cells, whereas much higher concentrations of SAMHD1 were detected in macaque alveolar macrophage, collected by bronchoalveolar lavage, and rhesus MDM derived in vitro. Cell lysates from the same preparations of freshly collected unstimulated rhesus macaque memory CD4+ T cells from blood or spleen were subjected to electrophoresis in Phos-tag Acrylamide and immunoblotting using the anti-SAMHD1 antibody. As shown in Fig 4B, most of the SAMHD1 in memory CD4+ T cells was unphosphorylated. The relatively high levels of expression of unphosphorylated endogenous SAMHD1 measured in memory CD4+ T lymphocytes suggested that SAMHD1 might significantly restrict viral infections in vivo, reduce virus production systemically, and affect disease progression if not adequately suppressed by Vpx.

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