Some positive-strand RNA plant viruses possess a transfer RNA-like structure
(TLS) at the 3'-terminus of their genomic RNAs. The closest mimicry to tRNA is
exhibited by the valylatable TLSs from tymoviruses and furo-like viruses, which are able
to interact with key cellular tRNA enzymes: [CTP, ATP]:tRNA nucleotidyltransferase
(CCA NTase), valyl-tRNA synthetase (ValRS), and translation elongation factor 1A
(eEF1A). In this thesis, I report the discovery of two new roles of the Turnip yellow
mosaic tymovirus TLS, in translation enhancement (Chapter 2) and repression of minus
strand initiation (Chapter 4).
Placement of the 3'-terminal 109 nts of TYMV RNA in a luciferase reporter RNA
with a generic 5'-UTR enhanced translation by about 20-fold in cowpea protoplasts.
Exhibiting a synergistic relationship with the 5'-cap, the 3'-translation enhancement was
largely dependent on the aminoacylatability of the TLS and apparently on eEF1A
interaction. In the presence of the 5'-UTR from genomic TYMV RNA, translation of
both the overlapping proteins p69 and p206 was strongly dependent on a 5'-cap structure,
and was enhanced by the 3'-enhancer. These in vivo results contradict the proposed
model in which translation initiation of p206, but not p69, is cap-independent and TLS-dependent (Barends et al. Cell 112(2003):123-9).
In vitro experiments with a partially purified preparation of TYMV replicase have
investigated the phenomenon of minus strand repression. Interaction of purified
eEF1A���GTP specifically with the valylated TLS decreased the template activity for
minus strand to near-background levels. eEF1A���GTP acts by making the 3'-CCA minus
strand initiation site unavailable to the replicase. The influence of eEF1A in
simultaneously enhancing translation and repressing minus strand synthesis can be
considered a regulation that ensures robust translation early in the infection and that
offers a coordinated transition from translation to replication.
Previously shown to be critical for TYMV infectivity, a valylatable TLS was
investigated for its role in the replication and infectivity of the bipartite Peanut clump
pecluvirus. A valylatable TLS provided a small competitive advantage in protoplasts and
whole plants. The advantage was more apparent in protoplasts than in whole plants, and
more so in the replication protein-encoding RNA1 than in the trans-replicating RNA2. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30728 |
| Date | 27 January 2004 |
| Creators | Matsuda, Daiki |
| Contributors | Dreher, Theo W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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