Doctoral Dissertations

Ruey-Yi Chang

Date of Award

12-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David A. Brian

Committee Members

Beth Mullin, W. Stuart Riggsby, David Hacker, Bert Lampson

Abstract

To study the cis-acting sequence requirements for coronavirus RNAreplication, a 2.2 kilobase defective-interfering (DI) RNA of the bovine coronavirus was cloned, sequenced, and produced synthetically so that it could be analyzed as a replicon after transfection into helper virus-infected cells. This experimental approach was necessary since efforts have not yet proven successful for cloning the 30 kilobase single-stranded RNAcoronavirus genome and synthesize it as an infectious molecule. The DIRNA is comprised of the genomic 5' and 3' termini of 498 and 1635 nucleotides (nt), respectively. Within the DI RNA, part of the polymerase and all of the nucleocapsid protein genes are fused together as a single open reading frame.

Through a systematic mutagenesis analysis of the cloned reporter- containing DI RNA, named pDrepl, this study has established the following points regarding coronavirus RNA replication: (1) Transfected reporter containing mRNA, differing from pDrep1 by only 433 nt between the 5' terminal 65 nt leader and the start of the N open reading frame, does not undergo replication as hypothesized earlier by our laboratory. Therefore, coronavirus mRNA species probably do not amplify by a replication mechanism and at least part of the 5' signal for RNA replication resides in the 433 nt unique sequence. (2) The 65 nt terminal leader that is common to the genome, DI RNA, and all subgenomic mRNA species, although not sufficient as a 5' terminal cis-acting signal, is a necessary part of the 5' terminal signal for RNA replication. This was demonstrated by terminal deletions of the leader for which it was shown that deletions of ≥ 13 nt, but not of ≤ 8 nt, were lethal for RNA replication. (3) When substitutions are made within the leader sequence, the 65 nt leader undergoes rapid reversion to the wild-type sequence during replication of pDrepl. This was demonstrated when a stem-loop of -8.0 kcal/mole between nucleotides 4 and 38, hypothesized to be an important part of the 5' cis- acting signal, was mutated to destroy the stem structure, and only wild- type leader was found in replicating molecules at 48 h post transfection. The phenomenon of rapid leader reversion may reflect the mechanism of free leader synthesis and a requirement for leader priming of replication. That is, there may be two steps in RNA replication: (a) abortive leader synthesis, and (b) leader priming of replication. (4) Most deletion and insertion mutations made within the unique 433 nt 5' terminal region of pDrep1 are lethal to RNA replication. Thus, most of the 5' terminal 498 nt are probably part of the 5' terminal signal for RNA replication. (5) Except for the 3' terminal 11% of the open reading frame, there is a strict requirement for maintaining the open reading frame in the replication of pDrep1. This was demonstrated by the loss of replicating ability in out-of- frame mutations made at 7 sites throughout pDrep. Differences in replication could not be explained by differences in mRNA stability since this was nearly the same for all constructs tested.

Thus, replication of coronavirus RNA, as determined by the study of DI RNA, requires an a unusually complex behavior of the leader and a requirement for maintaining an open reading frame. With this study, the coronavirus DI RNA has been established as a molecule with which to precisely determine many of the mechanisms involved in coronavirus plus and minus-strand RNA synthesis.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS