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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

Construction of the SIVmac239 and SIVmac316 X-del and X-Q76A Vpx mutants.(A) The codon (TCA) for the second residue (serine) of SIV Vpx was changed to a stop codon (TAA) to generate the X-del Vpx mutant. This change does not alter the amino acid sequence of the overlapping Vif protein. In the X-Q76A Vpx mutant, amino acid 76 (glutamine) was changed to alanine by altering the CAA codon to GCA. (B) The intracellular expression of Vpx proteins and their incorporation into virions was evaluated by transfecting HeLa cells with WT and Vpx mutant full-length SIV plasmids. Forty-eight hours later, cell lysates or cell-free culture supernatants, pelleted through 20% sucrose, were evaluated by immunoblotting using anti-SIV plasma, anti-Vpx antibodies, and anti-tubulin antibodies.
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ppat.1004928.g001: Construction of the SIVmac239 and SIVmac316 X-del and X-Q76A Vpx mutants.(A) The codon (TCA) for the second residue (serine) of SIV Vpx was changed to a stop codon (TAA) to generate the X-del Vpx mutant. This change does not alter the amino acid sequence of the overlapping Vif protein. In the X-Q76A Vpx mutant, amino acid 76 (glutamine) was changed to alanine by altering the CAA codon to GCA. (B) The intracellular expression of Vpx proteins and their incorporation into virions was evaluated by transfecting HeLa cells with WT and Vpx mutant full-length SIV plasmids. Forty-eight hours later, cell lysates or cell-free culture supernatants, pelleted through 20% sucrose, were evaluated by immunoblotting using anti-SIV plasma, anti-Vpx antibodies, and anti-tubulin antibodies.

Mentions: Several earlier studies have reported that replication competent HIV-2 and SIVmac mutants, unable to express the Vpx protein, exhibit delayed infection kinetics and low levels of progeny virus production in cultured rhesus peripheral blood mononuclear cells (PBMC) [9,14,15]. To ascertain whether a Vpx point mutant, specifically defective in recruiting DCAF1 and subsequently degrading SAMHD1[7,16–18], might possess similar properties, the X-Q76A Vpx mutant, carrying a 2-nucleotide substitution, was constructed (Fig 1A). A second Vpx mutant (X-del), containing a TAA stop codon at residue 2 that prevents the synthesis of any SIV Vpx protein was also prepared (Fig 1A). Both Vpx mutations were introduced into molecular clones of wild type (WT) T cell tropic (SIVmac239) or WT macrophage tropic (SIVmac316) SIVs. Wild type and Vpx mutant SIV inocula were prepared by transfecting full-length infectious molecular clones into 293T cells as described in Methods. Vpx expression in HeLa cells and its incorporation into progeny virions released into the transfection supernatant medium were confirmed by immunoblotting (Fig 1B).


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)

Construction of the SIVmac239 and SIVmac316 X-del and X-Q76A Vpx mutants.(A) The codon (TCA) for the second residue (serine) of SIV Vpx was changed to a stop codon (TAA) to generate the X-del Vpx mutant. This change does not alter the amino acid sequence of the overlapping Vif protein. In the X-Q76A Vpx mutant, amino acid 76 (glutamine) was changed to alanine by altering the CAA codon to GCA. (B) The intracellular expression of Vpx proteins and their incorporation into virions was evaluated by transfecting HeLa cells with WT and Vpx mutant full-length SIV plasmids. Forty-eight hours later, cell lysates or cell-free culture supernatants, pelleted through 20% sucrose, were evaluated by immunoblotting using anti-SIV plasma, anti-Vpx antibodies, and anti-tubulin antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004928.g001: Construction of the SIVmac239 and SIVmac316 X-del and X-Q76A Vpx mutants.(A) The codon (TCA) for the second residue (serine) of SIV Vpx was changed to a stop codon (TAA) to generate the X-del Vpx mutant. This change does not alter the amino acid sequence of the overlapping Vif protein. In the X-Q76A Vpx mutant, amino acid 76 (glutamine) was changed to alanine by altering the CAA codon to GCA. (B) The intracellular expression of Vpx proteins and their incorporation into virions was evaluated by transfecting HeLa cells with WT and Vpx mutant full-length SIV plasmids. Forty-eight hours later, cell lysates or cell-free culture supernatants, pelleted through 20% sucrose, were evaluated by immunoblotting using anti-SIV plasma, anti-Vpx antibodies, and anti-tubulin antibodies.
Mentions: Several earlier studies have reported that replication competent HIV-2 and SIVmac mutants, unable to express the Vpx protein, exhibit delayed infection kinetics and low levels of progeny virus production in cultured rhesus peripheral blood mononuclear cells (PBMC) [9,14,15]. To ascertain whether a Vpx point mutant, specifically defective in recruiting DCAF1 and subsequently degrading SAMHD1[7,16–18], might possess similar properties, the X-Q76A Vpx mutant, carrying a 2-nucleotide substitution, was constructed (Fig 1A). A second Vpx mutant (X-del), containing a TAA stop codon at residue 2 that prevents the synthesis of any SIV Vpx protein was also prepared (Fig 1A). Both Vpx mutations were introduced into molecular clones of wild type (WT) T cell tropic (SIVmac239) or WT macrophage tropic (SIVmac316) SIVs. Wild type and Vpx mutant SIV inocula were prepared by transfecting full-length infectious molecular clones into 293T cells as described in Methods. Vpx expression in HeLa cells and its incorporation into progeny virions released into the transfection supernatant medium were confirmed by immunoblotting (Fig 1B).

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