SAMHD1, a deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase), plays a key role in human innate immunity. It inhibits infection of blood cells by retroviruses, including HIV, and prevents the development of the autoinflammatory Aicardi-Goutières syndrome (AGS). The inactive apo-SAMHD1 interconverts between monomers and dimers, and in the presence of dGTP the protein assembles into catalytically active tetramers. Here, we present the crystal structure of the human tetrameric SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation via dGTP-induced tetramerization of two inactive dimers. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to yield the catalytically active enzyme. Structure-based biochemical and cell-based biological assays confirmed the proposed mechanism. The SAMHD1 tetramer structure provides the basis for a mechanistic understanding of its function in HIV restriction and the pathogenesis of AGS.The sterile alpha motif and HD-domain containing protein 1 (SAMHD1) dNTPase plays dual roles in human innate immunity. It restricts HIV-1 infection in immune cells of myeloid Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Accession codesThe coordinates and structure factors have been deposited in PDB, with accession code 4BZC for the wild type and 4BZB for the RN mutant. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript lineage and in quiescent CD4-positive T lymphocytes [1][2][3][4][5] . In these non-dividing cells, SAMHD1 reduces cellular dNTP levels to concentrations below the threshold required for reverse transcription of the viral RNA genome into DNA 6-8 . Furthermore, mutations in SAMHD1 are associated with an autoimmune condition, termed Aicardi Goutières Syndrome (AGS) 9,10 , whose clinical manifestations resemble congenital viral infection 11,12 . AGS-associated SAMHD1 mutations appear to disrupt the dNTPase activity of SAMHD1. Thus, SAMHD1's ability to negatively regulate cellular dNTP levels is essential for its roles in innate immunity 13,14 .The dNTPase activity of SAMHD1 resides in its histidine-aspartate (HD) domain, with the N-terminal sterile alpha motif (SAM) domain involved in other activities [13][14][15][16][17] . A recent crystal structure of a dimeric SAMHD1 catalytic core fragment (SAMHD1c1, residues 120-626) suggested an allosteric, dGTP-stimulated mechanism for the promotion of the dNTPase activity of dimeric SAMHD1 14 . However, the SAMHD1c1 structure did not contain substrate or the dGTP cofactor, thus providing limited insight into the mechanism of SAMHD1 activation. Recent biochemical and functional studies revealed that SAMHD1 interconverts between an inactive monomeric or dimeric form and a dGTP-induced tetr...

Background: SAMHD1, a dGTP-activated dNTPase, inhibits retrovirus infection at the reverse transcription step in monocytes and quiescent T lymphocytes.Results: dGTP-induced SAMHD1 tetramerization correlates with its functional activation.Conclusion: SAMHD1 tetramers are the biologically active form of this dNTPase.Significance: Learning how SAMHD1 function is regulated is important for understanding innate and anti-viral immunity.

Background: Human SAMHD1 protein restricts HIV/SIV infection of myeloid cells and is targeted for proteasomal degradation by HIV-2 Vpx protein.Results: Vpx binds the divergent C terminus of human SAMHD1 and loads it onto DCAF1 substrate receptor of CRL4 E3 ubiquitin ligase.Conclusion: Vpx programs SAMHD1 degradation by loading it onto CRL4DCAF1.Significance: Learning how viruses overcome innate anti-viral mechanisms is critical for the conception of new antiviral therapeutics.

Background: Human sterile ␣ motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase that is phosphorylated by cyclinA2-dependent kinases. Results: SAMHD1 mutants defective for cyclinA2 binding disrupt S phase progression, and this is alleviated by Thr-592 phosphomimetic mutation. Conclusion: CyclinA2-dependent kinases regulate SAMHD1 activity. Significance: SAMHD1 dNTP phosphohydrolase activity is regulated during the cell cycle.

NUT midline carcinoma (NMC) belongs to a class of highly lethal and poorly differentiated epithelial cancers arising mainly in human midline organs. NMC is caused by the chromosome translocation-mediated fusion of the NUT (nuclear protein in testis) gene on chromosome 15 to a few other genes, most frequently the BRD4 gene on chromosome 19. The mechanism by which the BRD4-NUT fusion product blocks NMC cellular differentiation and contributes to oncogenesis remains elusive. In this study, we show that BRD4-NUT and BRD4 colocalize in discrete nuclear foci that are hyperacetylated but transcriptionally inactive. BRD4-NUT recruits histone acetyltransferases to induce histone hyperacetylation in these chromatin foci, which provide docking sites for accumulation of additional BRD4 and associated P-TEFB (positive transcription elongation factor b) complexes in the transcriptionally inactive BRD4-NUT foci. These molecular events lead to repression of a BRD4⅐P-TEFB downstream target gene c-fos, a component of activator protein 1 (AP-1), that directly regulates epithelial differentiation. Knockdown of BRD4-NUT in NMC cells disperses the transcriptionally inactive chromatin foci and releases the transcriptional activators to stimulate c-fos expression, leading to restoration of cellular differentiation. Our study provides a novel mechanism by which the BRD4-NUT oncogene perturbs BRD4 functions to block cellular differentiation and to contribute to the oncogenic progression in the highly aggressive NMC.

The cellular bromodomain protein Brd4 is a major interacting partner of the papillomavirus (PV) E2 protein. Interaction of E2 with Brd4 contributes to viral episome maintenance. The E2-Brd4 interaction also plays an important role in repressing viral oncogene expression from the integrated viral genome in human PV (HPV)-positive cancer cells. However, the underlying mechanism is not clearly understood. In host cells, Brd4 recruits positive transcription elongation factor b (P-TEFb) to stimulate RNA polymerase II phosphorylation during cellular and viral gene expression. P-TEFb associates with the C terminus of Brd4, which largely overlaps with the E2 binding site on Brd4. In this study, we demonstrate that E2 binding to Brd4 inhibits the interaction of endogenous Brd4 and P-TEFb. P-TEFb is essential for viral oncogene E6/E7 transcription in both HeLa and CaSki cells that contain integrated HPV genomes. E2 binding to Brd4 abrogates the recruitment of P-TEFb to the integrated viral chromatin template, leading to inactivation of P-TEFb and repression of the viral oncogene E6/E7. Furthermore, dissociation of the Brd4-P-TEFb complex from the integrated viral chromatin template using a Brd4 bromodomain dominant-negative inhibitor also hampers HPV E6/E7 oncogene expression. Our data support that Brd4 recruitment of P-TEFb to the viral chromatin template is essential for viral oncogene expression. Abrogation of the interaction between P-TEFb and Brd4 thus provides a mechanism for E2-mediated repression of the viral oncogenes from the integrated viral genomes in cancer cells.Papillomaviruses (PVs) are small DNA tumor viruses that induce a variety of benign and malignant epithelial lesions in the infected host (for a review, see reference 29). Over 140 human PV (HPV) types have been identified to date. Depending on the potential for induction of malignant transformation, these viruses are further classified into high-risk and low-risk HPVs. High-risk HPVs are strongly associated with cervical cancer (69), which is the second most common cancer among women worldwide and the leading cause of death from cancer among women in developing countries.The PVs establish long-term, persistent infections. During latent infection, the viral genomes are maintained as extrachromosomal elements (episomes) that replicate along with host DNA. The PV E2 protein is a sequence-specific DNA binding protein involved in viral DNA replication, episome maintenance, and viral transcription (26). E2 consists of an N-terminal transcriptional activation domain linked to a C-terminal DNA binding/dimerization domain by a flexible hinge (39). The multiple functions of E2 rely on its sequence-specific recognition of a number of E2 binding sites within PV genomes. E2 initiates viral genome replication by loading the viral helicase E1 to the origin of replication (4, 43). It ensures the accurate partitioning of the replicated viral genomes to daughter cells by tethering them to host mitotic chromosomes (2,31,36,48,55). In addition, E2 can either activate or...

HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA “repair” inhibit HIV-1 infection.

Lentiviruses, including HIV-1, possess the ability to enter the nucleus through nuclear pore complexes and can infect interphase cells, including those actively replicating chromosomal DNA. Viral accessory proteins hijack host cell E3 enzymes to antagonize intrinsic defenses, and thereby provide a more permissive environment for virus replication. The HIV-1 Vpr accessory protein reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes and activates the DNA damage checkpoint in the G2 cell cycle phase. However, little is known about the roles played by these Vpr targets in HIV-1 replication. Here, using a sensitive pairwise replication competition assay, we show that Vpr endows HIV-1 with a strong replication advantage in activated primary CD4+ T cells and established T cell lines. This effect is disabled by a Vpr mutation that abolishes binding to CRL4DCAF1 E3, thereby disrupting Vpr antagonism of helicase-like transcription factor (HLTF) DNA helicase and other DNA repair pathway targets, and by another mutation that prevents induction of the G2 DNA damage checkpoint. Consistent with these findings, we also show that HLTF restricts HIV-1 replication, and that this restriction is antagonized by HIV-1 Vpr. Furthermore, our data imply that HIV-1 Vpr uses additional, yet to be identified mechanisms to facilitate HIV-1 replication in T cells. Overall, we demonstrate that multiple aspects of the cellular DNA repair machinery restrict HIV-1 replication in dividing T cells, the primary target of HIV-1 infection, and describe newly developed approaches to dissect key components.