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WIN 52035-dependent human rhinovirus 16: assembly deficiency caused by mutations near the canyon surface
1Institute for Molecular Virology, University of Wisconsin, Madison 53706-1596, USA.
Abstract:
Three drug-dependent mutants of human rhinovirus 16 (HRV16) were characterized by sequence analyses of spontaneous mutant isolates and were genetically reconstructed from a parental cDNA plasmid. These mutants formed plaques in the presence but not in the absence of the selecting antiviral drug, WIN 52035, which binds to the capsid of wild-type virus and inhibits its attachment to the host cell. The drug-dependent phenotype of each mutant was caused by a single amino acid substitution in the VP1 coat protein. The three independent mutations conferring drug dependence are M1103T, T1208A, and V1210A. Single-step growth experiments involving rescue of one of the three mutants (V1210A) by delayed drug addition suggested (i) that the drug dependence lesion is at the stage of virus assembly and (ii) that one or more components of the viral assembly pool decay in the absence of drug. RNA accumulation and infectivity were unaffected by the absence of drug in all three mutants, suggesting that the labile assembly component is coat protein.
Insights
Three drug-dependent human rhinovirus 16 mutants require the antiviral drug WIN 52035 for plaque formation. These mutations affect virus assembly, indicating a drug-dependent role for viral coat protein in this process.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- Human rhinovirus 16 (HRV16) is a common cause of the common cold.
- Antiviral drugs targeting viral capsid proteins can inhibit viral attachment and replication.
- Drug-dependent viral mutants present unique research opportunities for understanding viral mechanisms.
Purpose of the Study:
- To characterize the genetic basis and phenotypic behavior of drug-dependent HRV16 mutants.
- To investigate the role of the antiviral drug WIN 52035 in HRV16 replication and assembly.
- To identify the specific viral components affected by drug dependence.
Main Methods:
- Sequence analysis of spontaneous mutant isolates.
- Genetic reconstruction of mutants from a parental cDNA plasmid.
- Single-step growth experiments with delayed drug addition.
Main Results:
- Three drug-dependent HRV16 mutants were identified, each with a single amino acid substitution in the VP1 coat protein (M1103T, T1208A, V1210A).
- These mutants formed plaques only in the presence of WIN 52035.
- Drug dependence was localized to the virus assembly stage, with a labile component (likely coat protein) decaying in the absence of the drug.
Conclusions:
- Single amino acid substitutions in the VP1 coat protein can confer drug dependence to HRV16.
- The antiviral drug WIN 52035 is essential for the stability or function of a viral assembly component.
- These findings provide insights into the mechanisms of viral assembly and potential targets for antiviral therapies.