Structural and Functional Requirements for Homotypic and Heterotypic Interference by Indiana Serotype Defective Interfering Particles of Vesicular Stomatitis Virus
Bay, Pauline Hsiao Sing
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Permalink
https://hdl.handle.net/2142/77580
Description
Title
Structural and Functional Requirements for Homotypic and Heterotypic Interference by Indiana Serotype Defective Interfering Particles of Vesicular Stomatitis Virus
Author(s)
Bay, Pauline Hsiao Sing
Issue Date
1981
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
Ultraviolet light inactivation of Indiana serotype vesicular stomatitis virus and four defective interfering (DI) particles was measured to determine the target size for interference. In the case of DI particles whose genomes mapped at the 5' end of the virion RNA, this target size corresponded to the entire DI particle RNA molecule regardless of whether it amounted to 10%, 30%, or 50% of the viral genome. These data were interpreted as demonstrating that both termini of the DI particle RNAs were required for their replication and for interference with virion RNA replication.
In contrast, the unique heat-resistant (HR) DI particle, with an RNA molecule corresponding to the 3' half of the viral genome, exhibited an inactivation target size of approximately 42% of its RNA molecule with respect to both homotypic and heterotypic interference. Unlike other DI particles, this particle was subsequently found to interfere with primary transcription by homotypic and heterotypic virions. The unusual inactivation target size of the HR DI particle was thus interpreted as being a compromise between the requirements for replication of its genome and those for interference with virion primary transcription.
The ability of the HR DI particle to reduce virion primary transcription in vivo prompted the examination of DI particle interference in an in vitro transcription system. These experiments utilized viral ribonucleocapsids (RNCs) since the M protein of the virus appeared to inhibit transcription by virions. Only the exceptional HR DI RNC was found to interfere with the in vitro transcription by the Concan subtype of New Jersey serotype virions. Moreover, this ability was competitive both in vitro and in vivo and appeared to be dependent on the concentration ratios of DI particles to virions. This phenomenon was explained on the basis that the HR DI particle genome was capable of utilizing more polymerase molecules than were contained in the particle. The higher affinity for polymerase by the HR DI RNC was explicated in terms of dissociation events during transcription, which are presumably more frequent in the longer virion template. A mathematical expression consistent with the experimental results and based on these assumptions was derived.
The relevance of this interference with virion primary transcription to the lowering of viral yield from doubly infected cells was investigated by the construction of chimeric DI particles containing the HR DI particle genome with a thermolabile polymerase. At the nonpermissive temperature, these DI particles were unable to self-transcribe, interfere with virion primary transcription, or reduce virion yields but were able to be replicated. These results demonstrated that self-transcription of the HR DI particle genome was a prerequisite for heterotypic interference but not for its own replication. The correlation between inhibition with primary transcription and interference with viral production by HR DI particles was further supported by experiments which utilized the Hazelhurst subtype of New Jersey serotype virions. HR DI particle interference with primary transcription by these virions was observed only at those DI particle concentrations which were found to reduce virion yield.
With respect to the mechanism of DI particle interference, these data demonstrate that the HR DI particle interferes with virion yield via an alternate mechanism from the other DI particles. In this mechanism, inhibition of virion primary transcription is a prerequisite for interference with the yield of infectious progeny virus. However, events after this initial inhibition may be similar to those events for the other DI particles which map at the 5' terminus of the viral genome.
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