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Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation
R Sahli1, R Freund, T Dubensky
1Department of Pathology, Harvard Medical School, Boston, MA 02115.
Abstract:
We have examined effects of mutations of the minor capsid proteins VP2 and VP3 of polyoma virus. Infection of cells by mutants blocked in expression of either VP2 or VP3 by alterations of their initiator methionine codons led to the rapid selection of wild-type revertants. This reversion suggests a strong, if not absolute, requirement for both proteins in virus growth. A mutant virus, VP2*, in which alanine was substituted for glycine-2, expressed a nonmyristylated form of VP2 that was incorporated into virions. Studies of VP2* revealed a 15- to 20-fold lower specific infectivity and a delay in growth in both primary and established mouse cells compared to wild-type virus. Analysis of the growth delay indicated a defect in an early step of infection, at or prior to uncoating. Synthesis of viral DNA and VP1 was delayed by about 9 hr in mutant compared to that in wild-type infected cells. No evidence was obtained for an effect on either virus assembly (encapsidation) or stability of virions. Infection of mice by the VP2* mutant virus resulted in attenuated virus replication and tumor induction which were much more severely affected following intranasal inoculation than intraperitoneal inoculation.
Insights
Mutations in polyoma virus minor capsid proteins VP2 and VP3 severely impair viral growth and infectivity. A VP2 mutant showed delayed DNA replication and attenuated tumor induction in mice, highlighting the proteins essential roles.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Polyoma virus relies on capsid proteins for infection and replication.
- Minor capsid proteins VP2 and VP3 play roles in the viral life cycle.
- Understanding these proteins' functions is crucial for viral pathogenesis research.
Purpose of the Study:
- To investigate the functional significance of polyoma virus minor capsid proteins VP2 and VP3.
- To characterize a mutant virus with altered VP2 protein (VP2*) and its impact on viral infectivity and replication.
- To assess the in vivo effects of VP2 mutations on viral pathogenesis.
Main Methods:
- Site-directed mutagenesis to create mutants affecting VP2 and VP3 expression.
- Generation and characterization of a VP2 mutant (VP2*) with a nonmyristylated VP2 protein.
- In vitro infectivity assays in primary and established mouse cells.
- Analysis of viral DNA and VP1 protein synthesis kinetics.
- In vivo studies of viral replication and tumor induction in mice.
Main Results:
- Mutants deficient in VP2 or VP3 expression rapidly reverted to wild-type, indicating essential roles.
- The VP2* mutant exhibited 15- to 20-fold lower specific infectivity and delayed growth.
- A defect in an early infection step, likely uncoating, was observed for VP2*.
- Viral DNA and VP1 synthesis were delayed by approximately 9 hours in VP2* infected cells.
- VP2* infection led to attenuated viral replication and tumor induction in mice, particularly after intranasal inoculation.
Conclusions:
- Polyoma virus VP2 and VP3 proteins are essential for efficient viral growth and infectivity.
- Myristoylation of VP2 is important for optimal early infection steps, including uncoating.
- VP2 plays a critical role in viral DNA replication and pathogenesis.
- VP2 mutations lead to attenuated polyoma virus disease in vivo.