Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations.

Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV .

Structural disorder within paramyxovirus nucleoproteins and phosphoproteins.

This review focuses on the experimental data showing the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely Nipah (NiV), Hendra (HeV) and measles (MeV) viruses. We provide a detailed description of the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domains (N(TAIL)) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins. We also show that a significant flexibility persists within N(TAIL)-XD complexes, which therefore provide illustrative examples of "fuzziness".

Acyclic nucleoside thiophosphonates as potent inhibitors of HIV and HBV replication.

9-[2-(Thiophosphonomethoxy)ethyl]adenine 3 and (R)-9-[2-(Thiophosphonomethoxy)propyl]adenine 4 were synthesized as the first thiophosphonate nucleosides bearing a sulfur atom at the α-position of the acyclic nucleoside phosphonates PMEA and PMPA. Thiophosphonates S-PMEA 3 and S-PMPA 4 were evaluated for in vitro activity against HIV-1 (subtypes A to G), HIV-2 and HBV-infected cells, and found to exhibit potent antiretroviral activity. We showed that their diphosphate forms S-PMEApp 5 and S-PMPApp 6 are readily incorporated by wild-type (WT) HIV-1 RT into DNA and act as DNA chain terminators.

The N-terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA transcription.

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a 'cap-snatching' mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA.

Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses.

Structural disorder within Henipavirus nucleoprotein and phosphoprotein: from predictions to experimental assessment.

Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within alpha-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.

Background: To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties.

High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2'-5'-oligoadenylates.

Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2'-5' oligoadenylate synthetases, which synthesize 2'-5' adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2'-5' adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2'-5' adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail.

Structural disorder within the measles virus nucleoprotein and phosphoprotein.

In this review, we summarize the main experimental data showing the abundance of structural disorder within the measles virus (MeV) nucleoprotein (N) and phosphoprotein (P), and focus on the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domain of MeV N (N(TAIL)) upon binding to the C-terminal X domain of P (XD). The functional implications of structural disorder are discussed in light of the ability of disordered regions to establish a complex molecular partnership, thereby leading to a variety of biological effects, including tethering of the polymerase complex onto the nucleocapsid template, stimulation of viral transcription and replication, and virus assembly. We also discuss the ability of N(TAIL) to establish interactions with additional cellular co-factors, including the major inducible heat shock protein, which can modulate the strength of the N(TAIL)-XD interaction.

In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase.

Dengue virus replicons: production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity.

The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood.

The VIZIER project: overview; expectations; and achievements.

VIZIER is an acronym for a research project entitled "Comparative Structural Genomics of Viral Enzymes Involved in Replication" funded by the European Commission between November 1st, 2004 and April 30th, 2009. It involved 25 partners from 12 countries. In this paper, we describe the organization of the project and the culture created by its multidisciplinary essence.

Conformational disorder.

In recent years it was shown that a large number of proteins are either fully or partially disordered. Intrinsically disordered proteins are ubiquitary proteins that fulfill essential biological functions while lacking a stable 3D structure. Despite the large abundance of disorder, disordered regions are still poorly detected.

Synthesis and antiviral activity of boranophosphonate isosteres of AZT and d4T monophosphates.

We report synthesis, in vitro antiviral activity, and stability studies in biological media of original boranophosphonate isosteres of AZT and d4T monophophates. A convenient route for the synthesis of 3'-Azido-3'-deoxythymidine-5'-boranophosphonate 8 and 2',3'-Didehydro-3'-dideoxythymidine-5'-boranophosphonate 12 is described. H-phosphinates 7 and 11, and alpha-boranophosphonates 8 and 12 exhibited no significant in vitro activity against HIV-infected cells, neither against a broad panel of viruses, up to 200 microM.

High affinity binding between Hsp70 and the C-terminal domain of the measles virus nucleoprotein requires an Hsp40 co-chaperone.

The major inducible 70 kDa heat shock protein (hsp70) binds the measles virus (MeV) nucleocapsid with high affinity in an ATP-dependent manner, stimulating viral transcription and genome replication, and profoundly influencing virulence in mouse models of brain infection. Binding is mediated by two hydrophobic motifs (Box-2 and Box-3) located within the C-terminal domain (N(TAIL)) of the nucleocapsid protein, with N(TAIL) being an intrinsically disordered domain. The current work showed that high affinity hsp70 binding to N(TAIL) requires an hsp40 co-chaperone that interacts primarily with the hsp70 nucleotide binding domain (NBD) and displays no significant affinity for N(TAIL).

The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment.

Background: The genome of measles virus consists of a non-segmented single-stranded RNA molecule of negative polarity, which is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. The N protein possesses an intrinsically disordered C-terminal domain (aa 401-525, N(TAIL)) that is exposed at the surface of the viral nucleopcapsid. Thanks to its flexible nature, N(TAIL) interacts with several viral and cellular partners.

The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket.

Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.

Nucleocapsid structure and function.

Measles virus belongs to the Paramyxoviridae family within the Mononegavirales order. Its nonsegmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. This ribonucleoproteic complex is the substrate for both transcription and replication.

There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization.

In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.

MeDor: a metaserver for predicting protein disorder.

Background: We have previously shown that using multiple prediction methods improves the accuracy of disorder predictions. It is, however, a time-consuming procedure, since individual outputs of multiple predictions have to be retrieved, compared to each other and a comprehensive view of the results can only be obtained through a manual, fastidious, non-automated procedure. We herein describe a new web metaserver, MeDor, which allows fast, simultaneous analysis of a query sequence by multiple predictors and provides a graphical interface with a unified view of the outputs.

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.

The flavivirus polymerase as a target for drug discovery.

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication.

Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity.

The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation.