Progress 08/15/11 to 08/14/14
Outputs
Target Audience: This research may impact people in the field of molecular and cell biology with particular interest in the translation of proteins. Also, scientists in molecular plant virology may want to know the data presented. Changes/Problems: A difficulty encountered during this project was the isolation of the frameshifted protein in large quantity and high purity for mass spectrometry, which would allow us to determine the amino acid sequence across the frameshift site. This is crucial for determing whether the ribosome skips forward or backard during the frameshift event. Various tagging and expression methods are being explored to facilitate purification of this protein. What opportunities for training and professional development has the project provided? It allowed me to participate in an event consisting of the leading scientists studying alternative protein translation sponsored by a highly prestigeous organization, European Molecular Biology Organisation (EMBO). The EMBO workshop Recoding: Reprogramming genetic decoding was the most appropriate place to present the data to the most relevant audience. How have the results been disseminated to communities of interest? The data were orally reported to the assembly at the EMBO workshop Recoding: Reprogramming genetic recoding, in Killarney, Ireland, May 13-18, 2014, on the alternative methods of translation for a single RNA sequence. A seminar was given to the Interdepartmental Plant Biology major at Iowa State University. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Potyviridae consists of a third of all known plant viruses and cause disease worldwide. Individual and synergetic infections with other pathogens can lead to near total yield loss, 90% for various crops like potatos and stone fruits. Immediately after innoculation, these viruses have to manufacture viral proteins to establish infection. Understanding the mechanism of viral protein synthesis could potentially lead to an antiviral treatment. Potyviruses were recently discovered to use a the same coding sequence of nucleic acids to generate more than a single protein sequence. It is accomplished via alternative translation mechansims like frameshifting. Protein products will exist in a ratio that is optimized for viral proliferation. Perturbations to the specified ratio may compromise the viral life cycle leading to host resistance with a treatment to protect crops. Because viruses use host protein synthesis machinery, additional insight into translation of host proteins or general workings of the ribosome may be possible. Within the host, probable alternative translation produces a pool of previously unidentified proteins. Besides the characterization of the proteins, a novel working of the ribosome can be studied. The mechansim of frameshifting will provide additional information into the operation of ribosomes, a macromolecule conserved throughout all areas of life. The sequence of Turnip mosaic virus, a potyvirus, contains a G_GAA_AAA_A motif that facilitates the change in reading frame during translation of a viral polyprotein to allow translation of the small, overlapping gene. Normal translation of proteins has ribosomes reading the messenger RNA in increments of 3 bases called codons, but when a frameshift event occurs, the ribosome repositions itself to either reread or skip a number of bases. This causes the ribosome to now be out of sync with the previous reading frame denoted by directionality (+/-) and deviation (#). 1) To understand this frameshift mechanism, we performed characterization analysis of the sequences surrounding this viral motif using a dual luciferase reporter system. We found that the 8 base sequence alone can generate frameshifting, althrough the adjacent viral context does increase frameshift efficiency. No descrete RNA structural element was discovered within the sequence. This is unlike any other -1 programmed ribosomal frameshift signal due to the lack of structural element, and there are no known +2 frameshift signals. 2) Ambiguity of direction during the frameshift is currently being addressed by mass spectrometry analysis of the protein's amino acid sequence. By obtaining the protein during infection rather than an expression strategy, an accurate reflection of the ratio of proteins and directionality of the frameshift. 3) Using mutational analysis and sequences of viruses from every other genera within the Potyviridae family, frameshifting efficiency was found to be different for each genus and we found no consensus rate of frameshifting for the Potyviridae.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2015
Citation:
Hui, Alice Y. "ALternative Translation via Frameshifting Directed by an 8 Base Motif Found in Potyviridae." Thesis. Iowa State University, 2015. Print.
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Progress 08/15/12 to 08/14/13
Outputs
Target Audience: Molecular plant virologists interested in alternative translation mechanisms. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? On October 17, 2012, there was a panel discussion about effective and genuine networking which helped develop my professional and interpersonal skills. I attended the Seventh Biennial All Iowa Virology Symposium hosted by Iowa State University on March 29-30, 2013 to further my knowledge of the field of virology. The 2012 Walter E. and Helen Parke Loomis Distingushed Lecture and Plant Biology Mini-symposium on April 11-12 informed me of various methods and uses of plant imaging techniques. Later in April 2013, I facilitated an undergraduate student discussion about the scientific process during a yeast workshop that was part of a Howard Hughes Medical Institute (HHMI) training program. A teaching assistant training program in August 2013 helped prepare me to teach an undergraduate class for career exploration within a field of science. How have the results been disseminated to communities of interest? Experiments are still underway to obtain an accurate representation of the viral translation before dissemination. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, I will determine the frameshift signal of turnip mosaic potyvirus and potentially, the signal for viruses of other genera within Potyviridae to observe if the alternative translation mechanism is acquired through divergent or convergent development. This will be accomplished with the use of the dual luciferase constructs I created in this past period in both in vitro (wheat germ extract) and in vivo (protoplast from the maintained undifferentiated cultures of host species) as previously described. Because the predicted frameshift has only been proven with TuMV, results from this will not only allow comparison between genera of Potyviridae, but also experimentally validate the existence of the predicted overlapping open reading frame. The mutated motif in the infectious clone will be used in whole plant and protoplast to determine the impact of the motif and frameshifted P3NPIPO protein product upon infection and replication. It is predicted to have an effect on dissemination of the virus throughout the plant, but replication should proceed as normal. Use of the GST::P3 construct to produce enough protein for immunoblotting to detect the ratio of frameshifted, GSTP3NPIPO, to non-frameshfted, GST::P3, product to give the frameshift rate of TuMV during plant infection. The purified GST::P3NPIPO protein will then be used in mass spectrometry to identify the precise site and directionality of the frameshift, either -1 or +2. Directionality of the frameshift could potentially provide a brand new mechanism of frameshifting because there have not been any documented +2 frameshifts.
Impacts
What was accomplished under these goals?
To identify the exact site of frameshift, I generated a tagged P3NPIPO protein for purification and mass spectrometry. Glutathione (GST) is a common tag to assist in purifying proteins because there are readily available GST-specific affinity resins and columns. The tag was fused to the N-terminus of P3 because this allows the purification of both GST::P3 and GST::P3NPIPO from infected plant lysate to observe the ratio of non-frameshifted and frameshifted proteins, respectively. GST::P3NPIPO can be further isolated with electrophoresis and used for the mass spectrometry to determine the directionality of the ribosomal slippage of the frameshift. Constructs of the frameshift motif of representative viruses from the other genera of Potyviridae were generated to address aim 3. They consist of an upstream renilla luciferase gene connected to a downstream firefly luciferase gene via 200 bases of viral sequence. A total of 7 constructs, each representing a different genus besides Potyvirus, include blackberry virus Y for Brambyvirus, wheat yellow mosaic virus for Bymovirus, cucumber vein yellowing virus for Ipomovirus, Chinese yam necrotic mosaic virus for Macluravirus, sugarcane streak mosaic virus for Poacevirus, ryegrass mosaic virus for Rymovirus, and wheat streak mosaic virus for Tritimovirus. Comparison of the other genera to TuMV of the Potyvirus genus will provide data about the development of this alternative translation mechanism to assist in determining if the frameshift mechanism is similar across genera making it a characteristic of the family, frameshifting is specific to particular genera within Potyviridae, or convergent development of this alternative translation mechanism occurred. Cell cultures of various plant species were maintained for in vivo translation experiments. Oat, tobacco, and Arabidopsis calli have been propagated to facilitate generation of liquid suspension cultures. The liquid suspension cultures were used to produce protoplasts that are used as an in vivo environment to simulate the translation of viral proteins within the cells of a whole infected plant. Ongoing efforts have been made to produce an infectious clone of TuMV containing a mutate G2A6 motif. Difficulties of manipulating a large construct without undesired mutations have been and are being troubleshot. Understanding viral evolution and translational mechanisms may provide insight into future antiviral strategies or targets. The basic research may be applicable to viruses that infect animals or other organisms. Observing the virus manipulate the host cell machinery could allow scientists to battle pathogens in the ever evolving arms race against infection. This potentially leads to improved protection for our agricultural products and businesses in securing the food source of the future.
Publications
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Progress 08/15/11 to 08/14/12
Outputs
OUTPUTS: All plant viruses are parasites of the cell's proteins synthesis machinery (ribosomes). Viruses use non-canonical translation mechanisms to induce the ribosomes to synthesize viral proteins encoded in the viral mRNA. One such event is called ribosomal frameshifting. Frameshifting characterized to date, occurs when the ribosome changes reading frame during translation due to a signal comprised of a slippery sequence and a nearby downstream RNA structure. In contrast, the frameshift sequence in the RNA of potyviruses seems to differ from this consensus. To investigate the possible RNA structure that control potyvirus frameshifting, sequences surrounding the putative frameshift site, slippery sequence, of Turnip mosaic potyvirus (TuMV) were inserted between two luciferase reporter genes so that -1 frameshifting is required for expression of the downstream reporter. The ratio of reporter gene expression reflects the frameshift efficiency. Throughout the past year, 24 plasmid constructs were generated containing deletion and point mutations in the viral sequence, or with the sequence of Soybean mosaic potyvirus. These were tested both in vitro and in vivo in protein translation assays. Vectors were constructed for expression and purification of P3NPIPO, the frameshifted protein, to allow determination of the amino acid sequence at the frameshift site by mass spectrometry. This will determine the actual site of the frameshift. An Arabidopsis suspension cell culture system for rapid preparation of protoplasts, used in in vivo assays, was initiated as a platform to determine frameshift rate of constructs within a host plant of the virus. Dissemination: Findings have been shared at 17th Annual Meeting of the RNA Society in Ann Arbor, MI, May 2012 in poster format and at the USDA NIFA AFRI Pre/Postdoctoral PD meeting with a presentation. A discussion with collaborator Dr. Andrew Firth (Cambridge University, UK) occurred at the 31th Annual Meeting of American Society of Virology in Madison, WI. PARTICIPANTS: Individual: Alice Hui, Iowa State University, PD, W. Allen Miller, Iowa State University, mentor and co-PD. Collaborators: Andrew Firth, Cambridge University, bioinformatics and research advice, John Atkins, University of Utah, research advice. Training: Alice Hui, graduate student, Interdepartmental Plant Biology Major, Iowa State University. Alice is performing the work on ribosomal frameshifting. TARGET AUDIENCES: Plant virologists and molecular biologists. PROJECT MODIFICATIONS: The original proposal has identification of pause sites of ribosomes on the Turnip mosaic potyvirus genome as Aim 1a. The focus has changed to identification of ribosomal frameshift site by mass spectrometry of the protein product, P3NPIPO. This was not included in the proposal because a collaborator was working on this aspect of the project originally, but she has since stopped.
Impacts
Surprisingly, the sequence at the putative frameshift site was sufficient to produce significant frameshifting, about half that obtained when 200 bases surrounding the shift site were included. No specific RNA structure was needed, which is different from canonical -1 frameshifting. Hypothetically, frameshifting occurs anywhere the putative frameshift site, GGAAAAAA, exists, either in virus or plant genome. This new type of frameshift signal may provide an added layer of complexity to gene expression by expanding the potential of a single processed mRNA to encode multiple protein products. This information may suggest new antiviral strategies and it expands our knowledge about the coding capacity of cellular mRNAs in general.
Publications
- No publications reported this period
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