Xiaoyun Ji

Mechanism of Allosteric Activation of Samhd1 by Dgtp

SAMHD1, a dNTP triphosphohydrolase (dNTPase), has 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 through 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.

Structural basis of cellular dNTP regulation by SAMHD1.

Significance SAMHD1 is a dNTPase that depletes the cellular dNTP pool to inhibit the replication of retroviruses, including HIV-1. The dNTPase activity of SAMHD1 also enables the enzyme to be a major regulator of cellular dNTP levels in mammalian cells, in addition to be implicated in the pathogenesis of chronic lymphocytic leukemia (CLL) and Aicardi Goutières syndrome (AGS). Here we present extensive structural and enzymatic data to reveal how SAMHD1 is activated and regulated via the combined actions of GTP and all cellular dNTPs. Our work establishes a complete spectrum of nucleotide binding and the exquisite regulatory mechanism of SAMHD1 in cellular dNTP metabolism, retrovirus restriction, and the pathogenesis of CLL and AGS. The sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a dNTPase, prevents the infection of nondividing cells by retroviruses, including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with chronic lymphocytic leukemia (CLL) and the autoimmune condition Aicardi Goutières syndrome (AGS). The dNTPase activity of SAMHD1 can be regulated by dGTP, with which SAMHD1 assembles into catalytically active tetramers. Here we present extensive biochemical and structural data that reveal an exquisite activation mechanism of SAMHD1 via combined action of both GTP and dNTPs. We obtained 26 crystal structures of SAMHD1 in complex with different combinations of GTP and dNTP mixtures, which depict the full spectrum of GTP/dNTP binding at the eight allosteric and four catalytic sites of the SAMHD1 tetramer. Our data demonstrate how SAMHD1 is activated by binding of GTP or dGTP at allosteric site 1 and a dNTP of any type at allosteric site 2. Our enzymatic assays further reveal a robust regulatory mechanism of SAMHD1 activity, which bares resemblance to that of the ribonuclease reductase responsible for cellular dNTP production. These results establish a complete framework for a mechanistic understanding of the important functions of SAMHD1 in the regulation of cellular dNTP levels, as well as in HIV restriction and the pathogenesis of CLL and AGS.

Impaired dNTPase Activity of SAMHD1 by Phosphomimetic Mutation of Thr-592.

Background: Phosphorylation of SAMHD1 Thr-592 inhibits its anti-HIV activity. Results: Phosphomimetic mutation T592E of SAMHD1 perturbs SAMHD1 crystal structure, destabilizes SAMHD1 tetramer, and reduces its dNTP triphosphatase (dNTPase) activity. Conclusion: T592E decreases the dNTPase activity of SAMHD1 via destabilizing the catalytically active tetramer. Significance: Structure-induced impairment of SAMHD1 dNTPase activity by T592E suggests a mechanism of the phosphorylation-regulated SAMHD1 antiviral activity. SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutières syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by phosphorylation at residue Thr-592. The mechanism underlying the effect of phosphorylation on anti-HIV-1 activity remains unclear. SAMHD1 forms tetramers that possess deoxyribonucleotide triphosphate triphosphohydrolase (dNTPase) activity, which is allosterically controlled by the combined action of GTP and all four dNTPs. Here we demonstrate that the phosphomimetic mutation T592E reduces the stability of the SAMHD1 tetramer and the dNTPase activity of the enzyme. To better understand the underlying mechanisms, we determined the crystal structures of SAMHD1 variants T592E and T592V. Although the neutral substitution T592V does not perturb the structure, the charged T592E induces large conformational changes, likely triggered by electrostatic repulsion from a distinct negatively charged environment surrounding Thr-592. The phosphomimetic mutation results in a significant decrease in the population of active SAMHD1 tetramers, and hence the dNTPase activity is substantially decreased. These results provide a mechanistic understanding of how SAMHD1 phosphorylation at residue Thr-592 may modulate its cellular and antiviral functions.

The crystal structure of Zika virus helicase: basis for antiviral drug design.

The genus of Flavivirus contains important human pathogens, including dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses, which cause a number of serious human diseases throughout the world (Pierson TC, 2013). Zika virus (ZIKV) is also an arthropod-borne flavivirus, which was initially isolated in 1947 from a febrile sentinel rhesus monkey in the Zika forest in Entebbe, Uganda. ZIKV is transmitted by multiple Aedes mosquitoes (Lazear and Diamond, 2016). Historically, ZIKV infection typically caused a mild and self-limiting illness in human beings, accompanied by fever, headache, arthralgia, myalgia, and maculopapular rash (Ioos et al., 2014). ZIKV caught global attention in April 2007, when it caused a large epidemic of Asian genotype ZIKV in Yap Island and Guam, Micronesia. From 2013 to 2014, the Asian genotype was found responsible for the epidemics among several Pacific Islands, including French Polynesia, New Caledonia, Cook Islands, Tahiti, and Easter Island (Lazear and Diamond, 2016). In 2015, a rampant outbreak of ZIKV infection struck Brazil and other regions of the Americas, causing an estimated 1.3 million cases (Hennessey et al., 2016; Mlakar et al., 2016). Thereafter, ZIKV was found in fetal brain tissue, presumably accounting for the sharp increase of congenital microcephaly in the epidemic areas (Brasil et al., 2016; Mlakar et al., 2016; Rodrigues, 2016). Recent studies have demonstrated the significant cellular death of neural stem cells once infected with ZIKV, which provides direct evidence for the inhibitory role of ZIKV on fetal brain development (Tang et al., 2016). However, as there are currently no effective vaccines or therapies available to contain ZIKV infection, ZIKV remains a significant challenge to the public health of the Western Hemisphere as well as the whole world (Lazear and Diamond, 2016). Similar to other flaviviruses, ZIKV contains a singlestranded, positive sense RNA genome of 10.7 kb. The genome is translated into a single large polypeptide, which undergoes proteolytic cleavage into 3 structural proteins (C, prM/M, and E), and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Pierson TC, 2013). The NS3 protein is a key component for viral polypeptide processing and genomic replication, with a protease domain at its N-terminus and a helicase domain at the C-terminus. Upon stimulation by RNA, the helicase domain exhibits intrinsic nucleoside triphosphatase activity, which then provides the chemical energy to unwind viral RNA replication intermediates to facilitate replication of the viral genome together with RNA-dependent RNA polymerase (NS5) (Lindenbach, 2001). Given its essential role in genome replication, ZIKV helicase could be an attractive target for drug development against ZIKV. Here we report the crystal structure of ZIKV helicase at 1.8-Å resolution. The helicase structure revealed a conserved triphosphate pocket critical for nonspecific hydrolysis of nucleoside triphosphates across multiple flavivirus species. A positive-charged tunnel has been identified in the viral helicase, which is potentially responsible for accommodating the RNA. This crystal structure of ZIKV helicase provides an accurate model for rational drug design against ZIKV infection. We determined the crystal structure of ZIKV helicase at a resolution of 1.8 Å (Table S1) in the space group P21. Distinct from the DENV-2 helicase, whose two crystal forms both contain two molecules per asymmetric unit (Xu et al., 2005), ZIKV helicase has a solo protein molecule in an asymmetric unit in the crystals. No stable oligomer through crystallographic packing was identified in the crystals, consistent with the observation of a monomeric form of the ZIKV helicase in solution by size exclusion chromatography (Fig. 1A). This observation suggests that ZIKV helicase is able to function as a monomer. The refined model is complete and includes the residues 175–617 from ZIKV NS3. Although the overall structure is generally well ordered, the electron densities are less well defined for residues 193–202 and 249–255 with a higher B factor (>50 compared with an overall average B factor of 27). This indicates that these are possible substrate/ligand binding regions due to the increased flexibility. The tertiary structure of ZIKV helicase reveals three domains, of around 130–160 amino acid residues each (Fig. 1B and 1C). Domain I (residues 175–332) and domain II (residues 333–481) share a similar fold with an expanded six-stranded β-sheet stacked between a large number of loops and four helices, though there is little sequence identity between these two domains. Domain III (residues 482–617) is predominantly comprised of a four-α-

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

To the Editor: Sterile alpha motif domain– and HD domain–containing protein 1 (SAMHD1) is a cellular dNTP triphosphohydrolase (dNTPase) that restricts HIV-1 replication in myeloid cells and resting CD4+ T cells by degrading dNTPs and limiting viral reverse transcription1–5. Purified recombinant SAMHD1 also has exonuclease activity when synthetic nucleic acids or HIV-1 gag and tat RNAs transcribed in vitro are used as substrates6. Ryoo et al.7 recently suggested that SAMHD1 restricts HIV-1 infection through its ribonuclease (RNase) activity by cleaving the viral RNA genome. By using SAMHD1 mutants purported to specifically retain dNTPase (SAMHD1Q548A) or RNase (SAMHD1D137N) activities, Ryoo et al.7,8 proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in nondividing cells. They also suggested that SAMHD1 phosphorylation at T592 negatively regulated its RNase activity7. To extend these findings7, we measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-dependent HIV-1 reverse transcription was bypassed. We co-transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phosphoablative, but dNTPase-active, mutant (SAMHD1T592A; refs. 9–11) into human embryonic kidney (HEK) 293T cells and assessed intracellular HIV-1 Gag protein synthesis and viral particle release in the supernatants. This transfection-based HIV-1 production is independent of reverse transcription requiring intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA–mediated gene expression. Intracellular HIV-1 Gag protein levels, p24 capsid levels in released HIV-1 virions and infectivity were not reduced by the ectopic expression of WT SAMHD1 or SAMHD1T592A mutant (Supplementary Fig. 1), which suggests that SAMHD1 cannot inhibit HIV-1 production after the reverse transcription step, regardless of its phosphorylation at T592. These findings are consistent with our previous results showing that the expression of WT SAMHD1 or of the SAMHD1T592A mutant in dividing cells does not restrict HIV-1 infection11,12. Our results suggest that SAMHD1 does not have broad nuclease activity, but do not rule out a specific nucleolytic interaction between SAMHD1 and incoming HIV-1 genomic RNA (gRNA). Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, we reproduced the key experiments of Ryoo et al.7. Their conclusion that SAMHD1 restricts HIV-1 through RNase activity was based on the differential activity of the SAMHD1 mutants, SAMHD1D137N and SAMHD1Q548A (ref. 7). We independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure that there were no other disabling mutations in the constructs. We then examined the HIV-1 restriction and intracellular dNTP regulation by these mutants and WT SAMHD1 by following the protocol of Ryoo et al.7. Our results show that both the SAMHD1D137N and SAMHD1Q548A mutants were expressed at similar levels to that of WT SAMHD1, and each efficiently restricted HIV-1 infection and decreased dATP, dGTP and dTTP levels in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (Fig. 1a–c). SAMHD1 expression did not significantly decrease dCTP levels, as compared to vector control cells (Fig. 1c, right), probably owing to the different biosynthesis pathway of dCTP. Notably, Ryoo et al.7 showed only dCTP levels, but not dATP, dGTP or dTTP levels, in SAMHD1expressing or control cells. Previous studies have used a SAMHD1D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation and HIV-1 restriction of SAMHD1 (refs. 13–15). The SAMHD1D137A mutant has no detectable dNTPase activity or HIV-1 restriction in vitro, owing to its inability to form a stable tetramer14,15. It is possible that the SAMHD1D137N mutant might be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. It is also possible that an in vitro dNTPase assay using purified recombinant SAMHD1 proteins might not fully reflect the dNTPase activity of SAMHD1 in PMA-differentiated U937 cells. However, these possibilities remain to be examined to explain how the SAMHD1D137N mutant restricts HIV-1 infection if its dNTPase activity is impaired. In contrast to the results of Ryoo et al.7, the SAMHD1D137N and SAMHD1Q548A mutants in our experiments do not have differing anti-HIV-1 activities, and neither lacks the ability to lower cellular dNTP levels. Ryoo et al.7 reported an approximately twofold decrease in HIV-1 gRNA levels in PMA-differentiated U937 cells expressing WT SAMHD1 or SAMHD1D137N, but not the SAMHD1Q548A mutant, as compared to the control cells at 3 h and 6 h postinfection (h.p.i.), which suggests SAMHD1-mediated HIV-1 gRNA degradation7. By contrast, we detected comparable levels of HIV-1 gRNA in PMAdifferentiated U937 cells expressing SAMHD1 (WT, SAMHD1D137N and SAMHD1Q548A mutants) and the vector control cells at 1, 3 and 6 h.p.i., respectively (Fig. 1d), showing that, in our study, SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 h.p.i, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that the expression of WT SAMHD1, SAMHD1D137N and SAMHD1Q548A mutants significantly reduced HIV-1 late reverse transcription products as compared to the vector control cells (Fig. 1e), correlating well with a reduced intracellular dNTP pool (Fig. 1c). Thus, in our study, these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction. Seamon et al.16 reported that trace exonuclease activities of recombiSAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

Structural basis of Zika virus helicase in recognizing its substrates

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

A Mutation Identified in Neonatal Microcephaly Destabilizes Zika Virus NS1 Assembly in Vitro

An unprecedented epidemic of Zika virus (ZIKV) infection had spread to South and Central America. ZIKV infection was recently confirmed by CDC (the Centers for Disease Control and Prevention) to cause neonatal microcephaly, which posed a significant public health emergency of international concern. No specific vaccines or drugs are currently available to fight ZIKV infection. ZIKV nonstructural protein 1 (NS1) plays an essential role in viral replication and immune evasion. We determined the crystal structure of ZIKV NS1172–352, which forms a head-to-head, symmetric dimer with a unique 14-stranded β-ladder conserved among flaviviruses. The assembly of the β-ladder dimer is concentration dependent. Strikingly, one pathogenic mutation T233A (NCBI accession no. KU527068), found in the brain tissue of infected fetus with neonatal microcephaly, is located at the dimer interface. Thr233, a unique residue found in ZIKV but not in other flaviviruses, organizes a central hydrogen bonding network at NS1 dimer interface. Mutation of Thr233 to Ala disrupts this elaborated interaction network, and destabilizes the NS1 dimeric assembly in vitro. In addition, our structural comparison of epitopes for protective antibody 22NS1, targeting West Nile Virus NS1, could potentially be valuable in understanding its anti-virus specificities and in the development of antibodies against ZIKV.

Two Tales (Tails) of SAMHD1 Destruction by Vpx

The lentivirus protein Vpx/Vpr recognizes the host restriction factor SAMHD1 at either its N- or C-terminal tail and targets it for destruction by the cellular protein degradation machinery. In this issue of Cell Host & Microbe, Schwefel et al. (2015) report the structural basis of SAMHD1 N-terminal targeting by Vpx.