Progress 12/01/00 to 11/30/03
Outputs
The 3 prime untranslated region (UTR) is a master controller of BYDV RNA translation, subgenomic RNA (sgRNA) transcription, and genome replication. One sequence in the 3 prime UTR (the TE) of the viral genomic RNA confers cap-independent translation initiation at the 5 prime end of the viral genome and sgRNA1. We found we could engineer BYDV to express the heterologous Cereal yellow dwarf virus (CYDV) replicase via sgRNA1. This second replicase replicated different RNA sequences than BYDV, including a satellite RNA. We found that the sgRNA2 promoter extends from 50 nt upstream to 120 nt downstream of the 5 prime end of sgRNA2. Interestingly, the TE is inside the sgRNA2 promoter. To assess the relationship between TE and sgRNA2 promoter activities, mutations in the TE known to affect translation were tested for their effects on sgRNA2 promoter activity in oat protoplasts. None of the mutations known to affect TE-mediated cap-independent translation affected sgRNA2
synthesis. Thus, translation and sgRNA2 synthesis are functionally distinct. Another aim was to determine the role of small open reading frame 6 (ORF 6) which is predicted to be translated from sgRNA2. Using reporter gene fusions and epitope tags, we detected no translation product of sgRNA2 in cells, even though it translated in cell-free extracts. However, start codon or frameshift point mutations in ORF 6 knocked out virus replication, but did not affect cap-independent translation activity in vivo. We conclude that sequence in ORF 6 is a cis-acting RNA signal needed for replication but not translation. We surmise that sgRNA2 is unable to undergo the circularization that is necessary for in vivo translation of all mRNAs, because the TE is in the 5 prime UTR. In contrast, genomic RNA and sgRNA1 have sequences in their 5 prime UTRs that base pair to the TE in the 3 prime UTR allowing circularization. Instead of acting as an mRNA, we hypothesize that sgRNA2 is a trans-regulator of
translation, based on in vitro studies. We tested the effect of sgRNA2 on viral and host translation. By expressing the TE from unrelated Brome mosaic virus in oat protoplasts, we found that indeed the TE inhibits virus replication, and reporter gene translation, in trans. In contrast, using (i) flow cytometric sorting of infected cells harboring GFP-expressing virus, (ii) 1D and 2D gel electrophoresis, and (iii) transgenic expression of sgRNA2 in plants, we found no evidence that the TE or sgRNA2 globally inhibit host translation. This possibility still cannot be ruled out because TE expression levels may not have been high enough. Surprisingly, a point mutation that knocks out sgRNA2 synthesis, still permitted BYDV replication in plants. Thus, sgRNA2 is not essential for virus replication and movement in plants, but it may provide an advantage to the virus in subtle ways. We also discovered that Tobacco necrosis (TNV) has a BYDV-like TE, and poly(A) tail mimic sequences in its 3
prime UTR. We determined their secondary structures. TNV has a smaller genome than BYDV and it is mechanically transmissible, making it a good model for future studies.
Impacts
This basic research sheds light on the dynamic tension between RNA replication and translation that is common to all positive strand RNA viruses. The regulatory mechanisms of BYDV sgRNAs may be relevant to other viruses, such as SARS and Porcine Respiratory and Reproductive Syndrome virus (PRRS), that also produce numerous sgRNAs. The long-distance base pairing between the ends of the genome may inform studies of flaviviruses such as West Nile and dengue, which also require base pairing between ends of the genomic RNA. BYDV is much easier to work with and thus serves as a model system. This work on host-virus interactions also allows us to use BYDV RNA as a tool to help us understand how mRNAs in plants compete and interact with the protein synthesis apparatus. This in turn may assist in genetic engineering and other means of improving crops. Finally, BYDV is the most economically important virus of wheat, barley and oats, worldwide, and understanding how it works may
lead to novel means of controlling it.
Publications
- Song SI, Miller WA (2004) Cis and trans requirements for rolling circle replication of a satellite RNA. Journal of Virology 78, (In press).
- Allen AM, Miller WA (2004) Cap-independent and poly(A) tail-independent translation of plant viral RNAs. In "Viral Control of Gene Expression" K. Kean ed., Transworld Press, Kerala (In press).
- Shen R (2004) Gene regulation of RNA viruses with uncapped and non-polyadenylated genomic RNA. Ph.D. Dissertation, Genetics, Iowa State University (will result in three additional journal articles).
- Rakotondrafara AM (2002) The enigmatic role of ORF 6 of Barley yellow dwarf virus. M.S. Thesis, Plant Pathology, Iowa State University (will result in one journal article).
- Shen R, Miller WA (2004) The 3 prime untranslated region of Tobacco necrosis virus RNA contains a BYDV-like cap-independent translation element. Journal of Virology 78, (In press).
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Progress 01/01/02 to 12/31/02
Outputs
Control of BYDV gene expression by its 3' untranslated region (UTR) was investigated. The 3' cap-independent translation (protein synthesis) element (TE) is contained within the subgenomic (sg) RNA2 promoter. To determine whether the same RNA substructures that control cap-independent translation also control sgRNA2 synthesis, mutations in the TE with known effects on translation were introduced into a viral replicon and their effects on sgRNA2 synthesis were observed. Some substructures necessary for TE function were not necessary for sgRNA2 synthesis. Thus different factors probably interact with this sequence for the two functions. A sequence adjacent to the sgRNA3 promoter basepairs to a sequence 4 kb upstream to facilitate the ribosomal frameshifting necessary for translation of the viral polymerase. We propose that the long-distance base pairing needed for frameshifting and for translation initiation (last year's report) serves as a 'molecular traffic signal'
which allows the replicase proceeding from the 3' end to prevent translation far upstream by disrupting the base pairing. This would clear the viral RNA of ribosomes which would otherwise block the replicase and prevent replication. In the second part of the project, expression of TE-containing sgRNA2 was predicted to inhibit host translation as observed in vitro. None of the 24 transgenic lines (engineered with collaborator D. Somers, U. Minn.) showed significant expression of sgRNA2. This is expected if high levels of TE RNA are lethal due to translation inhibition. Translation from the abundant sgRNA2 in virus-infected, non-transgenic cells was not detectable using serological methods or by replacement of ORF 6 with a GFP gene. We predict that sgRNA2 lacks sequences needed for circularization of mRNA that is needed for translation in vivo. Mutations in and around the start codon of ORF6 are lethal to virus replication but did not affect cap-independent translation of genomic RNA.
We deduce that these bases, but not the encoded protein are necessary for virus replication. We conclude that sgRNA2 is a regulatory RNA rather than an mRNA. To further analyze the role of the TE, Tobacco necrosis virus (TNV) was used because its genome is 2 kb smaller than that of BYDV and unlike BYDV, it is mechanically transmissible and accumulates to high titers in plants. Deletion analysis of a full-length infectious TNV clone (from R. Coutts, Imperial College) revealed that a predicted BYDV-like 3' TE indeed confers cap-independent translation. TNV at least transiently infects Arabidopsis ecotype C24 plants, and the 3' TE's of both TNV and BYDV provide cap-independent translation of a reporter gene in Arabidopsis protoplasts. To test the effect on host translation, transgenic Arabidopsis plants were constructed that express sgRNA2 behind the estradiol-inducible UVX promoter (from N.-H. Chua, Rockefeller Univ.). No conclusions can be drawn at this early stage. The translation
experiments revealed that BYDV is more closely related to TNV (in a different virus family) than it is to other viruses in BYDV's family, suggesting that these viruses should be reclassified.
Impacts
This basic research may lead to (1) revision of taxonomic relationships in plant viruses which is important for epidemiology and possibly quarantine regulations, (2) more efficient engineering of plants for production of useful proteins, (3) understanding of control of replication by positive strand RNA viruses and thus better ways to prevent their occurrence. Some of this research has influenced researchers working with flaviviruses (e.g. West Nile, dengue, yellow fever), and possibly hepatitis C virus, which have similar RNA interactions to those of BYDV.
Publications
- Koev G, Liu S, Beckett R, Miller WA (2002) The 3'-terminal structure required for replication of barley yellow dwarf virus RNA contains an embedded 3' end. Virology 292:114-126.
- Atkins JF, Baranov PV, Fayet O, Herr AJ, Ivanov IP, Matsufuji S, Miller WA, Moore B, Prere MF, Wills NM, Jiadong Zhu J, Gesteland RF (2001)(published in 2002) Over-riding standard decoding enriches gene expression: Implications of recoding for ribosome function. In: Cold Spring Harbor Symposium on Quantitative Biology, vol. 66: The Ribosome. Cold Spring Harbor Laboratory Press, New York. pp. 217-232.
- Barry JK, Miller WA (2002) A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA. Proc Natl Acad Sci USA 99:11133-11138.
- Miller WA, Liu S, Beckett R (2002) Barley yellow dwarf virus: Luteoviridae or Tombusviridae? Molecular Plant Pathology 3:177-183.
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Progress 01/01/01 to 12/31/01
Outputs
Aim 1 is to identify structures on BYDV RNA that facilitate synthesis of viral subgenomic RNAs, and determine the mechanism(s) of this process. Previously we published the locations of the subgenomic RNA (sgRNA) promoters. This year we found additional enhancer sequences upstream of the sgRNA2 promoter. We also found, remarkably, that a short sequence near the sgRNA3 promoter base-pairs to a sequence 4 kilobases upstream to facilitate ribosomal frameshifting at the upstream site during translation of the viral replicase gene. Aim 2 tests a model of trans-regulation of translation of the viral mRNAs in which the cap-independent translation element (TE) in sgRNA2 specifically shuts off translation of genomic RNA while allowing translation of sgRNA1. We expressed the TE from an unrelated virus: brome mosaic virus (BMV). This construct, (but not a negative control with mutant TE) trans-inhibited BYDV replication in oat cells, most likely by inhibiting translation of
replicase from BYDV genomic RNA. Thus, our data show for the first time that the TE inhibits translation in trans in vivo. The TE in-cis stimulated expression of GFP in engineered BMV RNA and BMV 3a protein expression from BMV RNA. This was surprising because BMV RNAs are capped. To detect the effect of TE in trans on host translation, we infected protoplasts with GFP-expressing BYDV then separated infected from uninfected cells by automated fluorescent cell sorting and examined total synthesis of labeled proteins in each pool of cells. We could not detect a reliable difference in protein expression. This was confounded by the presence of numerous dead cells in the virus-negative pool. Thus, we cannot draw conclusions on the effect of BYDV or sgRNA2 on host gene expression. To examine the effect of the TE alone, we have engineered oats with TE-containing gene constructs. We are now screening the R0 oats for presence of transgene. We also investigated the existence of BYDV protein P6
which is encoded by sgRNA2. Using (1) antiserum against P6, (2) an epitope-tagged version of P6, (3) virus mutants lacking P6, and (4) a novel staining method specific for an amino acid motif in P6, we conclude that the protein product of ORF 6 is not expressed or is very unstable. This supports a role for sgRNA2 as a trans-regulator and not as an mRNA. It enhances but is not essential for virus replication in single cells.
Impacts
This research provides a better understanding of interactions of multiple RNAs with the plant protein synthesis apparatus, and in virus replication. While no direct applications are imminent from this basic research, it may lead to (1) new means of controlling the economically important BYDV and other viruses, (2) more efficient engineering of plants for production of useful proteins, and (3) a better understanding of any natural processes in plants that involve protein synthesis by multiple mRNAs.
Publications
- Guo L, Allen EM, Miller WA. 2001. Base-pairing between untranslated regions facilitates translation of an uncapped, non-polyadenylated viral mRNA. Molecular Cell 7:1103-1109.
- Paul CP, Barry JK, Dinesh-Kumar SP, Brault V, Miller WA. 2001. A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site. Journal of Molecular Biology 310:987-999.
- Baranov PV, Gurvich OL, Fayet O, Prere MF, Miller WA, Gesteland RF, Atkins JF, Giddings MC. 2001. RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression. Nucleic Acids Research 29:264-267.
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