Graduate Thesis Or Dissertation
 

Molecular characterization of infectious hematopoietic necrosis virus transcription and genome organization

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  • The transcription process of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus of salmonid fish, was studied both in vivo and in vitro. Polyadenylated RNA from IHNV infected-salmon cells was resolved electrophoretically into five bands, one of which was found to contain two mRNA species. The molecular weights and intracellular molar ratios of these bands were determined. Coding assignments were made by hybrid selection and in vitro translation of individual mRNA species using cloned plasmids carrying cDNA to each viral mRNA. Species of mRNA were identified which encode each of the five known virion proteins. In addition, a sixth viral mRNA species which encodes a previously unrecognized non-virion protein was discovered. This protein, designated NV, is the first non-virion protein reported for a rhabdovirus. Transcription by the RNA polymerase of IHNV was also examined in vitro. Studies of the optimal reaction conditions revealed that the use of HEPES buffer rather than Tris, and the addition of S-adenosyl methionine to the reactions led to a six-fold increase in enzyme activity. The products of in vitro transcription were found to contain polyadenylated species which co-migrated electrophoretically with IHNV mRNA bands 2, 3, 4, and 5 from IHNV infected-cells. These transcripts were shown to be functional mRNA species by the ability to direct the synthesis of viral proteins in vitro. The construction of cloned plasmids carrying cDNA to viral mRNA species is described. A set of 21 cloned plasmids was characterized by mRNA blot hybridizations and cross-hybridization studies, which identified subsets of plasmids with cDNA to each of the six viral mRNA species. The molecular weight of the genome RNA of IHNV was determined to be 3.7 x 10 ⁶. The cDNA clones were used to construct a physical map of the viral genome by heteroduplex analyses. Measurements of R-loops formed between viral genome RNA and cDNA cloned plasmids determined that the gene order on the viral genome is 3' N-Ml-M2-G-NV-L 5'.
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