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Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design. Author summary: Enteroviruses are responsible for a range of diseases. Virus assembly involves the cleavage of one of the capsid proteins, which is an absolute requirement for infectivity, however, is little understood. Here, we have applied molecular and computational approaches to trap a mutationally stabilised assembly intermediate virus particle. We have generated a high-resolution structure of this intermediate, and have proposed a model which defines the molecular mechanism of particle maturation and the role of the viral RNA genome in this process. Computationally applying our model to the broader family of picornaviruses supports the suggested mechanism, and further indicates that the cleavage uses an aspartic protease-like mechanism. The identification of these conserved features is likely to have implications for future vaccine and antiviral therapy design. [ABSTRACT FROM AUTHOR]

Structural studies of polio- and closely related viruses have provided a series of snapshots along their cell entry pathways. Based on the structures and related kinetic, biochemical, and genetic studies, [...]

The structure of the Ser 229 Ala mutant MurB was refined to 1.8 angstrom resolution, providing a clearer picture of the locations of key amino acid residues and solvent molecules involved in the catalytic reduction of MurB-enolpyruvyl-UDP-N-acetylglucosamine (EP-UDP-N-GlcNAc). The Ser 229 hydroxyl was found strategically placed from the C2 protonation site of EP-UDP-N-GlcNAc. An active-site H2O, Wat 500 which coordinates with Ser 229 for the protonation of C2 was also found. The loss of Ser OH was proven responsible for the disruption of H-bonding network in the reactions.

Both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc were proven to be substrates for UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli. Reduction of (E)-EB-UDP-GlcNAc with MurB in the presence of NADPD in D2O illustrated the stereochemistry of hydride addition at C3 and protonation at C2. The (E)-EB-UDP-GlcNAc was discovered to partition between reduction to UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site.

The replacement of the serine residue by the selenocysteine in the bacterial serine protease, substilisin, at 2.0-A resolution helps in deciphering the three-dimensional structure of selenosubtilisin, an artificial selenoenzyme. The selenium moiety occupies a position in the side chain of residue 221. Treatment of the enzyme with hydrogen peroxide oxidises it to seleninic acid, whose interactions with His64 and Asn155 in the oxyanion hole help in understanding the enzyme's its reactions with thiols and peroxides. Comparison of the structure of seneno subtilisin with glutathione peroxidase, a natural selenoenzyme, helps in the study of the role of the selenium prosthetic group.

A multiple copy simultaneous search (MCSS) program is used to design small combinatorial libraries of antipicornavirus capsid-binding ligands for poliovirus. It is shown that MCSS calculations correctly identified observed binding site for all the monomer units with the importance of ligand flexibility in the design process.