Identification and characterization ofcis-acting signals for RNA replication and transcription of vesicular stomatitis virus and its defective interfering particle

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Microbiology and Immunology

First Committee Member

Asit K. Pattnaik - Committee Chair


Transcription and replication of vesicular stomatitis virus (VSV) is initiated precisely from the 3$\sp\prime$-terminus of the genomic and antigenomic RNAs. To define and characterize the cis-acting signals that mediate these processes, the terminal sequences of VSV and one of its defective interfering (DI) particle genomes have been analyzed. A number of deletion and substitution mutants of DI cDNA were generated and the ability of the encoded mutant RNAs to be replicated was analyzed in the recombinant vaccinia virus-T7 RNA polymerase-based VSV RNA replication system. These studies have identified two nonoverlapping regions at the termini of the DI particle genome that play distinct roles in RNA replication. Region I, which encompasses nucleotides 1 to 24, is absolutely required for replication since DI RNA genomes lacking any part of this region were unable to replicate. Region II, spanning nucleotides 25 to 45, is not essential for replication but it functions as replication enhancer sequences (RES) in that the presence of these sequences confers high efficiency of replication to the template. In addition, deleting the sequences corresponding to the RES from both termini of DI RNA but maintaining the length of terminal complementarity as seen in the parental DI RNA resulted in templates that replicated poorly. Furthermore, when part or entire length of the RES was inserted or substituted into the 3$\sp\prime$-terminus of a VSV minigenome, the level of replication of the minigenome was increased at least 4-fold to as high as 15-fold. These results support the conclusion that specific sequences rather than the extent of terminal complementarity of DI RNA play a major role in efficient DI RNA replication. Based on these results, a model is proposed to account, in part, for the replicative dominance of DI RNA over the VSV genomic RNA.To examine the role of 3$\sp\prime$-terminal sequences of VSV in transcription and replication, a series of deletion mutants of a VSV minigenome was generated and the ability of the mutant minigenomes to be replicated and transcribed was analyzed. Results show that the 3$\sp\prime$-terminal nucleotides 1 to 18 are essential for VSV RNA replication. Transcription analysis indicates that the first 18 nucleotides are also required for transcription. Together, the results suggest that the cis-acting signals at the 3$\sp\prime$-terminus of VSV genome for transcription and replication may be overlapping.


Biology, Molecular; Biology, Microbiology

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