Bidirectional uncoating of the genomic RNA of a helical virus.

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.

Transcription termination at intrinsic terminators: the role of the RNA hairpin.

Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the lambda tR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNA-DNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.

Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins.