Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Salvatore Arrigo

Second Advisor

Michael G. Schmidt

Third Advisor

Joseph W. Dolan

Fourth Advisor

James Norris

Fifth Advisor

E.Carwile LeRoy

Abstract

The Human Immunodeficiency Virus Type 1 is a complex retrovirus, which produces a large number of proteins from a 10 Kb genome. The virus utilizes three overlapping open reading frames, a ribosomal frameshift event and three different classes of RNA (unspliced, partially spliced and fully spliced RNA) to perform this feat. To regulate the expression of these RNAs, HIV-1 requires Rev, a viral trans regulatory protein. The fully spliced RNAs are not dependent upon Rev for their expression. The unspliced and partially spliced RNAs are dependent on Rev to produce proteins. Rev binds to the Rev Response Element (RRE) located in the Rev dependent RNAs. The sequences responsible for the lack of production of protein in the absence of Rev, have been termed Cis-acting Repressor Sequences (CRS). Little is known about the mechanism or nature of CRS. It is believed that CRS serve to confer Rev dependence on the RNA and allow for the regulation of the RNA at a number of posttranscriptional levels. It is our hypothesis that, since Rev regulates the production of the different classes of mRNA, splice sites are intimately involved in the regulation of these RNAs by Rev. To define the role of Human Immunodeficiency Virus type 1 splice sites in the cytoplasmic accumulation of viral RNAs, sequential deletion mutagenesis on an infectious proviral clone of HIV-1 was performed. Deletion of the majority of intron sequences, containing previously identified CRS, did not attenuate CRS activity. Retention of either the first or second tat intron preserved CRS activity. RNAs containing splice donor sequences, in the absence of known downstream splice acceptor sequences, retained CRS activity. These results indicate that the major HIV-1 splice donors can function as CRS and function to negatively regulate the cytoplasmic accumulation of HIV-1 RNAs in COS cells. In order to further analyze the role of a splice donor in Rev dependence, the wild type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild type 5' splice donor, splicing still occurred, as measured by usage of the tat splice acceptor. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of RNA containing the cryptic splice donor demonstrated that Rev is still required for the cytoplasmic accumulation of the unspliced RNA, while spliced RNA is Rev independent. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor retains Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in determining the Rev-dependence of viral RNAs. These studies have investigated the critical role that splice sites can play in viral gene regulation and shown that splice sites, both wild-type and cryptic can function as CRS to control viral RNA expression.

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