Marcia Kremer

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS 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.

Structure determination of coxsackievirus B3 to 3.5 A resolution.

The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5 A resolution. The icosahedral CVB3 particles crystallize in the monoclinic space group, P2(1), (a = 574.6, b = 302.1, c = 521.6 A, beta = 107.7 degrees ) with two virions in the asymmetric unit giving 120-fold non-crystallographic redundancy. The crystals diffracted to 2.7 A resolution and the X-ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self-rotation function established pseudo R32 symmetry with each particle sitting on a 32 special position. This constrained the orientation and position of each particle in the monoclinic cell to near face-centered positions and allowed for a total of six possible monoclinic space-group settings. Correct interpretation of the high-resolution (3.0-3.2 A) self-rotation function was instrumental in determining the deviations from R32 orientations of the virus particles in the unit cell. Accurate particle orientations permitted the correct assignment of the crystal space-group setting amongst the six ambiguous possibilities and for the correct determination of particle positions. Real-space electron-density averaging and phase refinement, using human rhinovius 14 (HRV14) as an initial phasing model, have been carried out to 3.5 A resolution. The initial structural model has been built and refined to 3.5 A resolution using X-PLOR.

Transcription Factor YY1 Is a Vaccinia Virus Late Promoter Activator

Vaccinia virus has a DNA genome, yet replicates in the cytoplasmic compartment of the cell. We previously described the identification of a cellular protein having high affinity for vaccinia virus late promoter DNA. Sequence substitutions in the vaccinia I1L promoter were used to define a 5-nucleotide block at the transcription initiation site as essential for interaction with the protein. Within this sequence is the recognition motif for the nuclear transcription factor YY1. This factor regulates a multitude of cellular promoters, as an activator of transcription, as a repressor, or as an initiator element-binding protein. Antibodies directed against YY1 were used to show that YY1 copurified with the vaccinia late promoter-binding protein and was present in late promoter-protein complexes in gel supershift assays. Bacterially expressed YY1 also bound specifically to late promoter DNA. A dinucleotide replacement within the YY1 recognition motif directly adjacent to the transcription start site severely reduced the affinity of YY1 for the I1L promoter in vitro and impaired I1L promoter-dependent transcription in vivo. The intracellular localization of YY1 was shown by immunofluorescence microscopy to shift from primarily nuclear to the cytoplasm after vaccinia infection. These results indicate that YY1 has a positive role in the regulation of vaccinia virus late gene transcription and suggest that poxviruses have adapted cellular initiator elements as a means of regulating viral gene expression. This is the first identifiable cellular protein implicated in poxvirus transcription.

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.

Antiviral Activity of Distamycin A against Vaccinia Virus Is the Result of Inhibition of Postreplicative mRNA Synthesis

ABSTRACT Distamycin A has been described as an inhibitor of the cellular pathogenesis of vaccinia virus in culture. Distamycin is an antibiotic that specifically targets the minor groove of DNA. We show here that distamycin is a potent inhibitor of vaccinia virus replication. Pulse-labeling experiments showed that most major late proteins failed to accumulate in the presence of the antibiotic. We characterized the effect of distamycin on vaccinia virus nucleic acid biosynthesis with the goal of determining the inhibitors target. Early gene transcription was unaffected. DNA synthesis proceeded at normal rates, but DNA accumulated in large masses in the cytoplasm with no evidence of virion assembly. Transcription from the intermediate class promoter for the I1L gene was partially reduced by distamycin; however, transcription from the intermediate promoters for the three late transcription factor genes was severely inhibited. The accumulation of the late transcripts for the viral F17R and A10L genes also was severely impaired and was shown to be a direct inhibition of late promoter activity. These results indicate that inhibition of postreplicative intermediate and late transcription is the basis for inhibition of vaccinia virus by distamycin and indicate that DNA minor-groove ligands hold promise for effective anti-poxvirus drugs.

An In Vitro Transcription System for Studying Vaccinia Virus Early Genes

Transcription of the vaccinia virus early genes occurs within the confines of the virion core structure. Therefore, isolated virions are a particularly rich source of proteins that function in early mRNA biosynthesis. Methods are described here for the extraction of purified vaccinia virions to yield protein mixtures with high transcriptional activity on viral early gene templates, responding specifically to both transcriptional initiation and termination signals in the DNA.

Crystallographic and Pharmacological Studies of Antiviral Agents Against Human Rhinovirus

Picornaviruses are small RNA containing viruses that cause diseases in mammals such as polio, foot-and-mouth disease, and the common cold. For reasons not yet fully understood, some members of this family have a small number of serotypes while others have many (e.g. poliovirus has three serotypes while rhinovirus has 100 serotypes). One serotype is immunologically distinct from another such that an animal can become immune to one serotype but is still susceptible to infection by another. For this reason, vaccines have been developed to prevent poliovirus infection, but the great number of rhinovirus serotypes has thwarted the development of a rhinovirus vaccine. Therefore, in the case of rhinovirus, the only hope for a “cure” seems to lie in a pharmaceutical approach.

Viral Particles at Atomic Resolution

Crick and Watson (ref. 1) first recognized that spherical viruses had to be regular polyhedra. Of these, the icosahedron has the largest number (60) of asymmetric units and was subsequently found to be the preferred envelope. The coding capacity of the enclosed genetic material could therefore be limited to coding only a relatively limited structural protein(s) for one-sixth of the virion shell. The assembly of viral particles from smaller, repeated subunit s presents several defined advantages: such strategy of replication reduces considerably the amount of genetic information needed to code for the structural protein(s), and minimizes the risks of incurring in fatal errors, as faulty subunits inaccurately synthesized can be discarded at assembly time. The entire replication cycle of a virus, therefore, can be visualized as a two-step process: a) the synthesis of viral components (nucleic acid and proteins), i.e: a template dependent, energy consuming process of polymerization of preformed blocks (nucleotides or amino acids), and b) the self-assembly of the subunits into more complex structures, a process that does not involve the formation of stable chemical bonds but brings the the subunits (or the intermediate structures) to a thermodynamically stable configuration.