Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Dengue virus nonstructural protein 5 adopts multiple conformations in solution.

Dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a mRNA cap 1 structure ((7Me)GpppA(2'OMe)) at the 5' end of the viral genome and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, we conducted small-angle X-ray scattering experiments with the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms in which the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.

Full-length Dengue virus RNA-dependent RNA polymerase-RNA/DNA complexes: stoichiometries, intrinsic affinities, cooperativities, base, and conformational specificities.

Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and double-stranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ± 2 nucleotides. In the presence of Mg(2+), the site size increases to 29 ± 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex.

Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called 'B' as judged by the decrease in their inhibition potency when assayed with a 'B' site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the 'B' site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases.

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-A resolution of active coxsackievirus B3 RdRp (also named 3D(pol)) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D(pol) at this site excludes its uridylation by the carrier 3D(pol). We suggest that VPg at this position is either uridylated by another 3D(pol) molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D(pol)/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

Gln151 of HIV-1 reverse transcriptase acts as a steric gate towards clinically relevant acyclic phosphonate nucleotide analogues.

BACKGROUND: In the treatment of HIV, the loose active site of the HIV-1 reverse transcriptase (RT) allows numerous nucleotide analogues to act as proviral DNA 'chain-terminators'. Acyclic nucleotide phosphonate analogues (ANPs) represent a particular class of nucleotide analogue that does not possess a ribose moiety. The structural basis for their substrate efficiency regarding viral DNA polymerases is poorly understood. METHODS: Pre-steady-state kinetics on HIV-1 RT together with molecular modelling, were used to evaluate the relative characteristics of both the initial binding and incorporation into DNA of three different ANP diphosphates with progressively increasing steric demands on the acyclic linker: adefovir-diphosphate (DP), tenofovir-DP, and cidofovir-DP. RESULTS: The increase of steric demand in ANPs induced a proportional loss of the binding affinity to wild-type HIV-1 RT (Kd cidofovir-DP>>Kd tenofovir-DP>Kd adefovir-DP approximately Kd dNTPs), consistent with the lack of HIV-1 inhibitory activity for cidofovir. We show that, starting from adefovir-DP, the steric constraints mainly map to Gln151, as its mutation to alanine provides cidofovir-DP sensitivity. Interactions between the Gln151 residue and the methyl group of tenofovir-DP further increase with the mutation Gln151Met, resulting in a specific discrimination and low-level resistance to tenofovir-DP. This alteration is the result of a dual decrease in the binding affinity (Kd) and the catalytic rate (k(pol)) of incorporation of tenofovir-DP. By contrast, the tenofovir resistance mutation K65R induces a broad 'k(pol)-dependent' nonspecific discrimination towards the three ANPs. CONCLUSIONS: Overall, our results show that the efficiency of ANPs to compete against natural nucleotides as substrates for RT is determined by their close interaction with specific amino acids such as Gln151 within the RT active site. These results should help us to map and predict ANP sensitivity determinants in cellular and viral DNA polymerase active sites for which the understanding of different ANP sensitivity patterns are of medical importance.

A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus.

In (+) RNA coronaviruses, replication and transcription of the giant approximately 30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5'-(G/U)CC-3' trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

General catalytic deficiency of hepatitis C virus RNA polymerase with an S282T mutation and mutually exclusive resistance towards 2'-modified nucleotide analogues.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2'-C-methyl CTP (2'-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2'-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2'-modified nucleotide analogues. 2'-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2'-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2'-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

The modeled structure of the RNA dependent RNA polymerase of GBV-C virus suggests a role for motif E in Flaviviridae RNA polymerases.

BACKGROUND: The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp) plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV) is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV) is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C). Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. RESULTS: We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. CONCLUSION: Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA polymerases.

Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study.

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.