activation
48 The Identification of a novel posttranscriptional regulatory element in gamma retroviruses
Guy Pilkington1, Katarzyna Purzycka2, Jenifer Bear1, Stuart Le Grice2, Barbara Felber1
1Vaccine Branch, National Cancer Institute; 2Drug Resistance Program, National Cancer Institute
Posttranscriptional regulatory mechanisms of several simple retroviruses and retroelements have been elucidated with the only exception being the gamma retrovirus family, e.g. MuLV. We identified a novel ~1500 nt PostTranscriptional regulatory Element (PTE) located in pro-pol (nt 3184-4652) of MuLV and XMRV, being transferrable and promoting potent expression of an HIV gag reporter and the cat DM128 reporter transcript. Deletion of pro-pol and env from the provirus failed to produce Gag particles, whereas env deletion produced Gag particles, pointing to a critical role of the pro-pol sequence. We showed that the pro-polsequences could be replaced by heterologous RNA export elements e.g. CTE. We found that the RNA encoding γ-?retroviral gag only has a reduced half-life and this defect can be counteracted upon insertion of a pol or CTE elements acting in cis. SHAPE technology was applied to the pro-pol RNA, with particular emphasis on the PTE, which revealed a very complex stem-loop structure. Comparisons of pro-pol sequences from MuLV, XMRV and related γ-retroviruses showed that despite sequence divergence in the 3’ end of pol, their RNA structures show little variability, indicating that the RNA structure is invariant in PTE. We conclude that complex structural components are critical for function, in addition to sequences representing interaction sites for export factors. With our discovery of the PTE, we now have a complete picture of posttranscriptional regulation of simple retroviruses. Collectively, these viruses contain negative-acting sequences embedded in gag mediating RNA instability, hence impair expression. These viruses also contain positive-acting RNA elements, essential for promoting Gag expression. These RNA elements do not share sequence or structural features, their location within the full-length retroviral RNA can vary, but they depend on the cellular NXF1 export machinery.
RNA 2013 • Davos, Switzerland • June 11-16, 2013 Oral Abstracts
50 Inquiry into the variability of HCV IRES and its impact on function by developing and evaluation of a large-scale mutation database that also unfolds potential of some new nucleotides.
Anas Khawaja1, Vaclav Vopalensky1, Ludek Roznovsky2, Jakub Mrazek4, Ondrej Horvath3, Martin Pospisek1
1Charles University in Prague. Department of Genetics and Microbiology, Prague, Czech Republic;
2Infectious Diseases and AIDS Clinic, University Hospital Ostrava, Ostrava, Czech Republic; 3Institute of Molecular Genetics AS CR, Prague, Czech Republic; 4Institute of Public Health Ostrava, Ostrava, Czech Republic
Synthesis of the hepatitis C virus polyprotein is fully controlled by an IRES located within 5’UTR of the viral RNA. We developed a new flow-cytometry-based approach allowing us to monitor patient to patient differences in IRES activities of their whole viral populations. Simultaneously we employed more conventional methods comprising cloning of PCR fragments, DGGE, TGGE, sequencing and bicistronic reporter assay for finding new HCV IRES mutations and measuring their activities. Sequence data from patients’ samples along with analysis of migration patterns by DGGE and/or TGGE allowed us to estimate the variation that may have persisted over time within one or group of patients individually and collectively. We also compiled from the literature a comprehensive database comprising
~1300 IRES mutations further categorized by frequency, structural and functional behavior, experimental parameters, clinical data etc. Some IRESs bearing multiple mutations in various domains displayed either higher or lower levels of efficacy than expected after simple counting of known effects of the individual mutations. These differences in IRES activities raise the question of how some mutations are capable of sustaining an IRES WT activity. Could it be a long-range interaction between mutated nucleotides, and if yes, then how much is the extent of structure adaptability that IRES may need to go through for conservation of its function. We also collected data from published studies dealing with induced therapeutic response of interferon and ribavirin and its possible outcome in individuals with reference to occurrence of mutations in HCV IRES. By mapping out these mutations in sustained and non-sustained responders we observed no specific distribution that may conclude a direct impact in determining the therapy response between the two populations. We also illustrate impact of ~25 novel mutations found in our patients on the HCV function.
Oral Abstracts RNA 2013 • Davos, Switzerland • June 11-16, 2013
51 Novel Insights from Structural Analysis of Lentiviral and Gammaretroviral Reverse Transcriptases in Complex with RNA/DNA Hybrids.
Stuart Le Grice1, Mikalai Lapkouski7, Lan Tian6, Jennifer Miller1, Enzbieta Nowak4, Wojciech Potrzebowski3, Peter Konarev2, Jason Rausch1, Marion Bona1, Dmitri Svergun2, Janusz Bujnick3, Marcin Nowotny4, Wei Yang5
1HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA; 2European Molecular Biology Laboratory, Hamburg, Germany; 3Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular & Cell Biology, Warsaw, Poland.; 4Laboratory of Protein Structure, International Institute of Molecular & Cell Biology, Warsaw, Poland.; 5National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA; 6National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA; 7National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/
DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid1. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented.
Although the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species.
We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV)2. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.
1. Lapkouski, M., Tian, L., Miller, J.T., Le Grice, S.F.J., and Yang, W. (2013). Complexes of HIV-1 RT, an NNRTI and an RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat. Struct. Mol. Biol. 20: 230-236.
2. Nowak, E., Potrzebowski, W., Konarev, P.V., Rausch, J.W., Bona, M.K., Svergun, D.I., Bujnicki, J.M., Le Grice, S.F.J., and Nowotny, M. (2013). Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res., in press.
RNA 2013 • Davos, Switzerland • June 11-16, 2013 Oral Abstracts