David C. Goldstone

HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase

SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA

There are two distinct pathways for disulfide formation in prokaryotes. The DsbA‐DsbB pathway introduces disulfide bonds de novo, while the DsbC‐DsbD pathway functions to isomerize disulfides. One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo. Cross‐talk between these two systems would be mutually destructive To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation. In these mutants, DsbC is present as a monomer as compared with dimeric wild‐type DsbC. Based on these findings we rationally designed DsbC mutants in the dimerization domain. All of these mutants are able to rescue the dsbA null phenotype. Rescue depends on the presence of DsbB, the native re‐oxidant of DsbA, both in vivo and in vitro. Our results suggest that dimerization acts to protect DsbCs active sites from DsbB‐mediated oxidation. These results explain how oxidative and reductive pathways can co‐exist in the periplasm of Escherichia coli.

The disulfide bond isomerase DsbC is activated by an immunoglobulin‐fold thiol oxidoreductase: crystal structure of the DsbC–DsbDα complex

The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N‐terminal domain (DsbDα) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC–DsbDα complex. The 2.3 Å crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDα is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V‐shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDα reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDα active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.

Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein-protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer-hexamer spacing characteristic of the viral core is produced.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice.

Significance Retroviral infection of cells can be blocked by the action of the postentry restriction factors. The Trim5α and Fv1 factors do so by targeting the capsid that surrounds the viral core. The nature of the interaction of these factors with the viral assembly is unclear. We show that these factors form antiparallel dimers that display specificity domains spaced to target motifs on the capsid lattice surface. In doing so Fv1 and Trim5α take advantage of the regularly spaced array of binding sites on the capsid surface, generating avidity to aid recognition of retroviral pathogens. Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.

Novel Escape Mutants Suggest an Extensive TRIM5α Binding Site Spanning the Entire Outer Surface of the Murine Leukemia Virus Capsid Protein

After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5α. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5α and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer ‘top’ surface of N-MLV CA, including the N-terminal β-hairpin, and map up to 29 Ao apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5α, indicating significant differences in the binding interaction between N-MLV and the two TRIM5αs, whereas three of the mutations result in dual sensitivity to Fv1n and Fv1b. Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5α binding.

Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

Structural and Functional Analysis of Prehistoric Lentiviruses Uncovers an Ancient Molecular Interface.

Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution.

Structure of the RNA15 Rrm-RNA Complex Reveals the Molecular Basis of Gu Specificity in Transcriptional 3-End Processing Factors.

Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3′-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3′-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3′-end processing complexes that interact with the U-rich or U/G-rich elements at 3′-end cleavage/polyadenylation sites.

Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo‐form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA‐bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant‐derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin‐3‐carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized. Proteins 2010.