Mutations in the zinc-binding motif of the reovirus capsid protein delta 3 eliminate its ability to associate with capsid protein mu 1.

Reovirus capsid protein delta 3 binds both double-stranded RNA (dsRNA) and zinc. Previous studies have revealed that the amino-terminal zinc finger is not required for the ability of delta 3 to bind dsRNA. We expressed wild-type and mutant delta 3 molecules by in vitro transcription/translation to evaluate the importance of the zinc finger for other functions of delta 3. delta 3 molecules with mutations in the zinc finger did not form complexes with capsid protein mu 1 but bound dsRNA more efficiently than wild-type delta 3 did. In contrast, a dsRNA-binding mutant was unimpaired in its ability to associate with mu 1. Studies with delta 3 fragments support these findings and indicate that sequences critical for delta 3's interaction with mu 1 lie in the amino terminus of the molecule. Our finding that mu 1 and dsRNA do not compete for identical binding sites on delta 3 has implications for its function as a translational regulator in infected cells.

Association of reovirus outer capsid proteins sigma 3 and mu 1 causes a conformational change that renders sigma 3 protease sensitive.

Association of the reovirus proteins sigma 3 and mu 1 influences viral entry, initiation of outer capsid assembly, and modulation of the effect of sigma 3 on cellular translation. In this study, we have addressed whether structural changes occur in sigma 3 as a result of its interaction with mu 1. Using differences in protease sensitivity to detect conformationally distinct forms of sigma 3, we showed that association of sigma 3 with mu 1 caused a conformational change in sigma 3 that converted it from a protease-resistant to a protease-sensitive structure and occurred posttranslationally. The effect of mu 1 on the structure of sigma 3 was stoichiometric. Our results are consistent with a model in which sigma 3's association with mu 1 shifts its function from translational control to assembly of an outer capsid in which sigma 3 is folded into the protease-sensitive conformation that is required for its cleavage during the next round of infection.

Use of drug-resistance mutants to identify functional regions in picornavirus capsid proteins.

The WIN drugs and similar hydrophobic compounds that insert into the capsid of picornaviruses have been shown to block viral uncoating. In some of the human rhinoviruses they also block attachment of virus to cells. Spontaneously occurring drug-resistant mutants of human rhinovirus 14 and poliovirus type 3 were selected for their ability to make plaques in the presence of the selecting drug. The HRV-14 mutants either prevented drug binding or allowed the virus to attach to cells in the presence of drug. About two thirds of the poliovirus mutants were dependent on the presence of drug for plaque formation. In single cycle growth curves, drug was not required for the formation of drug-dependent progeny virus. However, progeny virus grown without drug never accumulated outside of cells, thus making the formation of plaques impossible. This behavior was apparently caused by the extreme thermolability of these mutants. In the absence of drug, heating to 37 degrees C rapidly converted them to non-infectious particles with a sedimentation coefficient of 135S.

WIN 52035-2 inhibits both attachment and eclipse of human rhinovirus 14.

WIN compounds inhibit attachment of human rhinovirus 14 by binding to a hydrophobic pocket within the capsid and inducing conformational changes in the canyon floor, the region that binds the cellular receptor. To study the basis of drug resistance, we isolated and characterized a family of human rhinovirus 14 mutants resistant to WIN 52035-2. Thermostabilization data and single-cycle growth curves provided evidence for two classes of resistant mutants. One class, here called exclusion mutants, showed a marked decrease in drug-binding affinity and was characterized by substitution to bulkier amino acid side chains at two sites lining the hydrophobic pocket. The other class, called compensation mutants, displayed single-amino-acid substitutions in the drug-deformable regions of the canyon; these mutants were able to attach to cells despite the presence of bound drug. A delay in the rise period of the growth curves of compensation mutants indicated a second locus of drug action. WIN 52035-2 was found to inhibit the first step of uncoating, release of VP4. Attempts to identify this site of drug action by using single-step growth curves were obscured by abortive elution of a major fraction of cell-attached virus. The drug had no effect on the rate of this process but did affect the spectrum of particles produced.

Genetic and molecular analyses of spontaneous mutants of human rhinovirus 14 that are resistant to an antiviral compound.

Spontaneous mutants of human rhinovirus 14 resistant to WIN 52084, an antiviral compound that inhibits attachment to cells, were isolated by selecting plaques that developed when wild-type virus was plated in the presence of high (2 micrograms/ml) or low (0.1 to 0.4 micrograms/ml) concentrations of the compound. Two classes of drug resistance were observed: a high-resistance (HR) class with a frequency of about 4 x 10(-5), and a low-resistance (LR) class with a 10- to 30-fold-higher frequency. The RNA genomes of 56 HR mutants and 13 LR mutants were sequenced in regions encoding the drug-binding site. The HR mutations mapped to only 2 of the 16 amino acid residues that form the walls of the drug-binding pocket. The side chains of these two residues point directly into the pocket and were invariably replaced by bulkier groups. These findings, and patterns of resistance to related WIN compounds, support the concept that HR mutations may hinder the entry or seating of drug within the binding pocket. In contrast, all of the LR mutations mapped to portions of the polypeptide chain near the canyon floor that move when the drug is inserted. Because several LR mutations partially reverse the attachment-inhibiting effect of WIN compounds, these mutants provide useful tools for studying the regions of the capsid structure involved in attachment. This paper shows that the method of escape mutant analysis, previously used to identify antibody binding sites on human rhinovirus 14, is also applicable to analysis of antiviral drug activity.