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2.
Proc Natl Acad Sci U S A ; 110(42): E3997-4006, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-24082141

ABSTRACT

Macrophages are a major target cell for HIV-1, and their infection contributes to HIV pathogenesis. We have previously shown that the cyclin-dependent kinase inhibitor p21 inhibits the replication of HIV-1 and other primate lentiviruses in human monocyte-derived macrophages by impairing reverse transcription of the viral genome. In the attempt to understand the p21-mediated restriction mechanisms, we found that p21 impairs HIV-1 and simian immunodeficiency virus (SIV)mac reverse transcription in macrophages by reducing the intracellular deoxyribonucleotide (dNTP) pool to levels below those required for viral cDNA synthesis by a SAM domain and HD domain-containing protein 1 (SAMHD1)-independent pathway. We found that p21 blocks dNTP biosynthesis by down-regulating the expression of the RNR2 subunit of ribonucleotide reductase, an enzyme essential for the reduction of ribonucleotides to dNTP. p21 inhibits RNR2 transcription by repressing E2F1 transcription factor, its transcriptional activator. Our findings unravel a cellular pathway that restricts HIV-1 and other primate lentiviruses by affecting dNTP synthesis, thereby pointing to new potential cellular targets for anti-HIV therapeutic strategies.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Deoxyribonucleotides/biosynthesis , Gene Expression Regulation, Enzymologic , HIV Infections/metabolism , HIV-1/physiology , Macrophages/metabolism , Ribonucleotide Reductases/biosynthesis , Virus Replication/physiology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , DNA, Viral/biosynthesis , DNA, Viral/genetics , Deoxyribonucleotides/genetics , Down-Regulation/genetics , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , HIV Infections/therapy , HIV Infections/virology , Macrophages/virology , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Ribonucleotide Reductases/genetics , SAM Domain and HD Domain-Containing Protein 1 , Simian Immunodeficiency Virus/physiology , Transcription, Genetic/genetics
3.
PLoS Pathog ; 9(6): e1003481, 2013.
Article in English | MEDLINE | ID: mdl-23825958

ABSTRACT

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.


Subject(s)
Herpesvirus 1, Human/physiology , Monomeric GTP-Binding Proteins/metabolism , Myeloid Cells/metabolism , Myeloid Cells/virology , Vaccinia virus/physiology , Virus Replication/physiology , Cell Line , Female , Humans , Male , Monomeric GTP-Binding Proteins/genetics , Myeloid Cells/pathology , SAM Domain and HD Domain-Containing Protein 1
4.
J Biol Chem ; 288(35): 25001-25006, 2013 Aug 30.
Article in English | MEDLINE | ID: mdl-23880768

ABSTRACT

SAMHD1 (SAM domain- and HD domain-containing protein 1) is a dGTP-dependent dNTP triphosphohydrolase that converts dNTPs into deoxyribonucleosides and triphosphates. Therefore, SAMHD1 expression, particularly in non-dividing cells, can restrict retroviral infections such as HIV and simian immunodeficiency virus by limiting cellular dNTPs, which are essential for reverse transcription. It has previously been established that dGTP acts as both an activator and a substrate of this enzyme, suggesting that phosphohydrolase activity of SAMHD1 is regulated by dGTP availability in the cell. However, we now demonstrate biochemically that the NTP GTP is equally capable of activating SAMHD1, but GTP is not hydrolyzed by the enzyme. Activation of SAMHD1 phosphohydrolase activity was tested under physiological concentrations of dGTP or GTP found in either dividing or non-dividing cells. Because GTP is 1000-fold more abundant than dGTP in cells, GTP was able to activate the enzyme to a greater extent than dGTP, suggesting that GTP is the primary activator of SAMHD1. Finally, we show that SAMHD1 has the ability to hydrolyze base-modified nucleotides, indicating that the active site of SAMHD1 is not restrictive to such modifications, and is capable of regulating the levels of non-canonical dNTPs such as dUTP. This study provides further insights into the regulation of SAMHD1 with regard to allosteric activation and active site specificity.


Subject(s)
Guanosine Triphosphate/chemistry , Monomeric GTP-Binding Proteins/chemistry , Deoxyguanine Nucleotides/chemistry , Deoxyguanine Nucleotides/genetics , Deoxyguanine Nucleotides/metabolism , Enzyme Activation/physiology , Guanosine Triphosphate/genetics , Guanosine Triphosphate/metabolism , HIV/genetics , HIV/metabolism , Humans , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , SAM Domain and HD Domain-Containing Protein 1
5.
J Biol Chem ; 288(28): 20683-91, 2013 Jul 12.
Article in English | MEDLINE | ID: mdl-23744077

ABSTRACT

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.


Subject(s)
Deoxyribonucleosides/metabolism , HIV-1/drug effects , Monomeric GTP-Binding Proteins/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Blotting, Western , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , HIV-1/genetics , HIV-1/physiology , Host-Pathogen Interactions , Humans , Macrophages/drug effects , Macrophages/metabolism , Macrophages/virology , Monomeric GTP-Binding Proteins/genetics , Nevirapine/pharmacology , RNA Interference , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/physiology , Virion/drug effects , Virion/genetics , Virion/physiology , Virus Replication/drug effects , Zidovudine/pharmacology
6.
Retrovirology ; 10: 23, 2013 Feb 25.
Article in English | MEDLINE | ID: mdl-23442224

ABSTRACT

BACKGROUND: Type I interferon (IFN) treatment of some cells, including dendritic cells, macrophages and monocytic THP-1 cells, restricts HIV-1 infection and prevents viral cDNA accumulation. Sterile alpha motif and HD domain protein 1 (SAMHD1), a dGTP-regulated deoxynucleotide triphosphohydrolase, reduces HIV-1 infectivity in myeloid cells, likely by limiting dNTPs available for reverse transcription, and has been described as IFNα-inducible. Myeloid cell infection by HIV-1 is enhanced by HIV-2/SIVSM Vpx, which promotes SAMHD1 degradation, or by exogenous deoxyribonucleoside (dN) addition. FINDINGS: SAMHD1 expression was not substantially influenced by IFNα treatment of monocyte-derived macrophages or THP-1 cells. The contributions of SAMHD1 to the inhibition of HIV-1 infectivity by IFNα were assessed through the provision of Vpx, exogenous dN addition, or via RNAi-mediated SAMHD1 knock-down. Both Vpx and dN efficiently restored infection in IFNα-treated macrophages, albeit not to the levels seen with these treatments in the absence of IFNα. Similarly using differentiated THP-1 cells, the addition of Vpx or dNs, or SAMHD1 knock-down, also stimulated infection, but failing to match the levels observed without IFNα. Neither Vpx addition nor SAMHD1 knock-down reversed the IFNα-induced blocks to HIV-1 infection seen in dividing U87-MG or THP-1 cells. Therefore, altered SAMHD1 expression or function cannot account for the IFNα-induced restriction to HIV-1 infection seen in many cells and cell lines. CONCLUSION: IFNα establishes an anti-HIV-1 phenotype in many cell types, and appears to accomplish this without potentiating SAMHD1 function. We conclude that additional IFNα-induced suppressors of the early stages of HIV-1 infection await identification.


Subject(s)
HIV-1/immunology , Interferon-alpha/immunology , Macrophages/immunology , Macrophages/virology , Monomeric GTP-Binding Proteins/metabolism , Cell Line , Gene Knockdown Techniques , Humans , Monomeric GTP-Binding Proteins/genetics , Nucleotides/metabolism , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/metabolism
7.
Virology ; 436(2): 247-54, 2013 Feb 20.
Article in English | MEDLINE | ID: mdl-23260109

ABSTRACT

Retroviruses consume cellular deoxynucleoside triphosphates (dNTPs) to convert their RNA genomes into proviral DNA through reverse transcription. While all retroviruses replicate in dividing cells, lentiviruses uniquely replicate in nondividing cells such as macrophages. Importantly, dNTP levels in nondividing cells are extremely low, compared to dividing cells. Indeed, a recently discovered anti-HIV/SIV restriction factor, SAMHD1, which is a dNTP triphosphohydrolase, is responsible for the limited dNTP pool of nondividing cells. Lentiviral reverse transcriptases (RT) uniquely stay functional even at the low dNTP concentrations in nondividing cells. Interestingly, Vpx of HIV-2/SIVsm proteosomally degrades SAMHD1, which elevates cellular dNTP pools and accelerates lentiviral replication in nondividing cells. These Vpx-encoding lentiviruses rapidly replicate in nondividing cells by encoding both highly functional RTs and Vpx. Here, we discuss a series of mechanistic and virological studies that have contributed to conceptually linking cellular dNTP levels and the adaptation of lentiviral replication in nondividing cells.


Subject(s)
Cytoplasm/chemistry , Lentivirus/physiology , Nucleotides/metabolism , Retroviridae/physiology , Reverse Transcription , Virus Replication , Animals , Humans , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/analysis , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/metabolism
8.
Retrovirology ; 9: 105, 2012 Dec 11.
Article in English | MEDLINE | ID: mdl-23231760

ABSTRACT

BACKGROUND: SAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown. RESULTS: Here, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool. CONCLUSIONS: Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Dendritic Cells/metabolism , Dendritic Cells/virology , HIV-1/physiology , Monomeric GTP-Binding Proteins/metabolism , CD4-Positive T-Lymphocytes/drug effects , Dendritic Cells/drug effects , Gene Expression Regulation/drug effects , HEK293 Cells , HeLa Cells , Humans , Interferons/pharmacology , Monomeric GTP-Binding Proteins/genetics , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcription , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/metabolism , Virus Replication
9.
J Biol Chem ; 287(17): 14280-8, 2012 Apr 20.
Article in English | MEDLINE | ID: mdl-22383524

ABSTRACT

Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.


Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Reverse Transcriptase/metabolism , Macrophages/virology , Reverse Transcription/physiology , Base Sequence , DNA Repair , DNA Replication , HIV-1/genetics , HIV-1/metabolism , Humans , Hydrolysis , Jurkat Cells , Kinetics , Macrophages/cytology , Molecular Sequence Data , Nucleotides/genetics , Ribonuclease H/metabolism , Ribonucleotides/genetics , Time Factors
10.
Biochemistry ; 48(20): 4285-93, 2009 May 26.
Article in English | MEDLINE | ID: mdl-19338266

ABSTRACT

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.


Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , G-Protein-Coupled Receptor Kinase 2/physiology , Animals , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Crystallography, X-Ray/methods , Humans , Kinetics , Models, Molecular , Molecular Conformation , Peptides/chemistry , Phosphorylation , Protein Structure, Tertiary , Receptors, Adrenergic, beta-2/metabolism , Rhodopsin/chemistry
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