The C2 domain acts as a membrane‐targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium‐dependent interactions with phosphatidylserine. The three‐dimensional structures of the Ca2+‐bound forms of the PKCα‐C2 domain both in the absence and presence of 1,2‐dicaproyl‐ sn ‐phosphatidyl‐L‐serine have now been determined by X‐ray crystallography at 2.4 and 2.6 Å resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi‐cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca2+ ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca2+ binding region. The central feature of the PKCα‐C2 domain structure is an eight‐stranded, anti‐parallel β‐barrel with a molecular topology and organization of the Ca2+ binding region closely related to that found in PKCβ‐C2, although only two Ca2+ ions have been located bound to the PKCα‐C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCα‐C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH₂-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template–primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in ( i ) the recognition and positioning of the incoming nucleotide or analog; ( ii ) the positioning of the acceptor base of the template strand; and ( iii ) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.