The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

The bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase can initiate RNA replication by a de novo mechanism without a primer. The structure of BVDV polymerase, determined to 2.9-Å resolution, contains a unique N-terminal domain, in addition to the fingers, palm, and thumb domains common to other polymerases. The structure of BVDV polymerase complexed with GTP, which is required for de novo (primer-independent) initiation, shows that GTP binds adjacent to the initiation NTP, suggesting that the GTP mimics a vestigial RNA product. Comparison of five monomers in two different crystal forms showed conformational changes in the fingertip region and in the thumb domain that may help to translocate the RNA template and product strands during elongation. The putative binding sites of previously reported BVDV inhibitors are also discussed.T he function of a polymerase is to synthesize the complementary RNA or DNA molecule from a template strand. Thus, the polymerase requires binding sites for the template, nucleoside triphosphates (NTPs or dNTPs), and the nascent polynucleotide product. Also required are mechanisms to catalyze the addition of NTP or dNTP to the nascent chain, to recognize the next complementary NTP molecule, and to move the template and product by one nucleotide in readiness for the next elongation event. Finally, the polymerase, in conjunction with other viral and cellular enzymes, such as a helicase in a replication complex, requires a mechanism for initiating and terminating synthesis of the new product polynucleotide.RNA replication in positive-sense single-stranded (ss)RNA viruses is initiated at or near the 3Ј end of the template using either primer-dependent or primer-independent (de novo) mechanisms. Poliovirus, for example, utilizes a genome-linked protein as a primer for initiation of RNA synthesis (1). In Flaviviridae, on the other hand, de novo initiation is the likely mechanism used during virus replication in infected cells (2). In de novo initiation, the second NTP is added (with the release of its pyrophosphate moiety) directly to the 3Ј-OH of the first initiation NTP without the need of a primer. This nucleotidyl transfer reaction is then repeated with subsequent NTPs to generate the complementary RNA product. De novo initiation by Flaviviridae RNA polymerases requires (i) a template RNA with a virus-specific initiation nucleotide at the 3Ј end, (ii) a complementary initiation NTP, and (iii) GTP (3-6). Because base pairing between the template RNA and individual NTPs may not be sufficient to allow the formation of a stable initiation complex, it is likely that de novo initiation requires specific molecular interactions among the template RNA, NTPs, the RNA polymerase, and possibly other viral and host proteins. Although not all RNA-dependent RNA polymerases (RdRps) require GTP for initiating RNA synthesis, other ligands or structural components can act similarly as GTP and help to position the 3Ј-OH group of the priming nucleotide ready for nucleophilic attack.The Flavivirida...

The activity of pleconaril in cell culture against prototypic enterovirus strains and 215 clinical isolates of the most commonly isolated enterovirus serotypes was examined. The latter viruses were isolated by the Centers for Disease Control and Prevention during the 1970s and 1980s from clinically ill subjects. Pleconaril at a concentration of ≤0.03 μM inhibited the replication of 50% of all clinical isolates tested. Ninety percent of the isolates were inhibited at a drug concentration of ≤0.18 μM. The most sensitive serotype, echovirus serotype 11, was also the most prevalent enterovirus in the United States from 1970 to 1983. Pleconaril was further tested for oral activity in three animal models of lethal enterovirus infection: coxsackievirus serotype A9 infection in suckling mice, coxsackievirus serotype A21 strain Kenny infection in weanling mice, and coxsackievirus serotype B3 strain M infection in adult mice. Treatment with pleconaril increased the survival rate in all three models for both prophylactic and therapeutic dosing regimens. Moreover, pleconaril dramatically reduced virus levels in target tissues of coxsackievirus serotype B3 strain M-infected animals. Pleconaril represents a promising new drug candidate for potential use in the treatment of human enteroviral infections.

Pleconaril (VP 63843) is a novel orally bioavailable small molecule with broad antipicornavirus (enterovirus and rhinovirus) activity. Ten independently derived pleconaril-resistant variants of coxsackievirus B3 were isolated from cell culture. The molecular basis of drug resistance and the biologic properties of the drug-resistant viruses were investigated. RNA sequence analysis revealed amino acid changes in the drug-binding pocket of the resistant variants. Thermal stability studies showed the drug-resistant viruses to be significantly less stable than wild type virus. When evaluated in a murine model in which wild type virus infection is 100% lethal, the drug-resistant viruses showed attenuated virulence with both reduced mortality and delayed time to death. Virus titers in heart and spleen were dramatically lower in drug-resistant virus-infected mice than in wild type virus-infected animals. The study results indicate that pleconaril-resistant virus variants are attenuated and significantly less virulent than drug-sensitive wild type virus.

Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.