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1.
Sci Rep ; 6: 26616, 2016 05 27.
Article in English | MEDLINE | ID: mdl-27229711

ABSTRACT

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.


Subject(s)
Hepatitis B virus/physiology , Hepatocytes , Mutation, Missense , SAM Domain and HD Domain-Containing Protein 1/metabolism , Virus Replication/physiology , Amino Acid Substitution , Hep G2 Cells , Hepatocytes/enzymology , Hepatocytes/pathology , Hepatocytes/virology , Humans , SAM Domain and HD Domain-Containing Protein 1/genetics
2.
PLoS Pathog ; 7(12): e1002425, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22174685

ABSTRACT

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.


Subject(s)
Autoimmune Diseases of the Nervous System/genetics , Genetic Predisposition to Disease/genetics , HIV Infections/genetics , Monomeric GTP-Binding Proteins/deficiency , Monomeric GTP-Binding Proteins/genetics , Myeloid Cells/virology , Nervous System Malformations/genetics , Autoimmune Diseases of the Nervous System/metabolism , Autoimmune Diseases of the Nervous System/virology , Cell Separation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , HIV-1/metabolism , Humans , Immunoblotting , Immunoprecipitation , Lipopolysaccharide Receptors/metabolism , Microscopy, Confocal , Mutation, Missense , Myeloid Cells/metabolism , Nervous System Malformations/metabolism , Nervous System Malformations/virology , Reverse Transcriptase Polymerase Chain Reaction , SAM Domain and HD Domain-Containing Protein 1 , Tandem Mass Spectrometry , Transfection
3.
Viruses ; 3(7): 1112-30, 2011 07.
Article in English | MEDLINE | ID: mdl-21994773

ABSTRACT

Virus infections elicit an immediate innate response involving antiviral factors. The activities of some of these factors are, in turn, blocked by viral countermeasures. The ensuing battle between the host and the viruses is crucial for determining whether the virus establishes a foothold and/or induces adaptive immune responses. A comprehensive systems-level understanding of the repertoire of anti-viral effectors in the context of these immediate virus-host responses would provide significant advantages in devising novel strategies to interfere with the initial establishment of infections. Recent efforts to identify cellular factors in a comprehensive and unbiased manner, using genome-wide siRNA screens and other systems biology "omics" methodologies, have revealed several potential anti-viral effectors for viruses like Human immunodeficiency virus type 1 (HIV-1), Hepatitis C virus (HCV), West Nile virus (WNV), and influenza virus. This review describes the discovery of novel viral restriction factors and discusses how the integration of different methods in systems biology can be used to more comprehensively identify the intimate interactions of viruses and the cellular innate resistance.


Subject(s)
Immunity, Innate/immunology , Systems Biology/methods , Virus Diseases/immunology , Viruses/immunology , Genomics/methods , Host-Pathogen Interactions/immunology , Humans , Virus Diseases/virology
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