Viral and Subviral RNA Replication and Translation

We are studying cap-independent translation of two viruses:  Turnip crinkle virus (TCV), Pea enation mosaic virus (PEMV) and Saguaro cactus virus (SCV).  Both viruses contain 3' cap-independent translational enhancers in their 3' regions that represent new paradigms for translation.  Our lab uses state-of-the-art RNA structure mapping (low and high throughput), single-molecule biophysical techniques such as optical tweezers, and genetic manipulations to elucidate how 3' proximal ribosome binding elements enhance translation.

TCV:  We have discovered that a 3’ translational enhancer in Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) composed of three hairpins and two pseudoknots that binds to 60S ribosomal subunits in the P-site.  TCV does not contain any discernable long-distance RNA:RNA interactions, but rather ribosomes appear to "bridge" the 5' and 3' ends of the genome by binding simultaneously to 5' and 3' sequences.  Interactions in the 3'UTR and upstream coding region are extraordinarily complex, and are likely needed for ribosome recruitment, ribosome recycling, and the switch from translation to replication when sufficient RdRp has been synthesized. 

PEMV:  PEMV contains translation elements throughout its 3' UTR including a 3' terminal hairpin that engages in a long-distance interaction with the upstream ribosome recoding hairpin, 3' proximal TSS, a PTE-type translation enhance, the adjacent kl-TSS that binds to ribosomes and can simultaneously engage in a long-distance interaction with a coding region hairpin near the 5' end, and a phylogentically conserved upstream hairpin. 

Selected Publications

Dashi, A., Schwander, P., Langlois, R., Fung, R., Li, W., Hosseinizadeh, A.,Liao, H. Y., Pallesen, J., Sharma, G., Stupina, V.A., Simon, A.E., Dinman, J.D., Frank, J., Ourmazd, A.  Trajectories of the ribosome as a Brownian nanomachine.  Proc. Natl. Acad. Sci. USA, in press.

Gao, F., Reddy, S., Kasprzak, W., Shapiro, B.A., Dinman, J.D., and Simon, A.E.  2014. The kl-TSS translational enhancer of PEMV can bind simultaneously to ribosomes and a 5’ proximal hairpin.  J. Virol. 88, 11696–11712.

Chattopadhyay, M., Kuhlmann, M., Kumar, K., and Simon, A.E.  2014.  Position of the kissing-loop interaction associated with PTE-type 3’CITEs can affect enhancement of cap-independent translation.  Virology, 458-459, 43-52.

 Simon, A. E., and Miller, W. A. 2013. 3’ Cap-independent translation enhancers of plant viruses. Annu. Rev. Microbiol. 67: 21–42.

Stupina, V.A. and Simon, A.E.  2013.   Preparation of biologically active Arabidopsis ribosomes and comparison with yeast ribosomes for binding to a tRNA-mimic that enhances translation of plant plus-strand RNA viruses. Front. Plant Sci. 4, 271. doi: 10.3389/fpls.2013.

Gao, F., Kasprzak, W., Stupina, V.A., Shapiro, B.A. and Simon, A.E.  2012.  A ribosome-binding, 3’ translational enhancer has a T-shaped structure and engages in a long distance RNA:RNA interaction. J. Virol. 86, 9828-9842.

Yuan, X., Shi, K., and Simon, A.E.  (2012).  An interactive network of RNA elements supports translation and replication in Turnip crinkle virus.  J. Virol. 86, 4065-4081.

Guo, R., Meskauskas, A., Dinman, J.D., and Simon, A.E.  (2011).  Evolution of a helper virus-derived ribosome binding translational enhancer in an untranslated satellite RNA of Turnip crinkle virus.  Virology 419, 10-16.

Chattopadhyay, M., Shi, K., Yuan, X., and Simon, A.E.  (2011).  Long-distance kissing loop interactions between a 3’ proximal Y-shaped structure and apical loops of 5’ hairpins enhance translation of Saguaro cactus virus.  Virology 417, 113-125.

Stupina, V.A, Yuan, X, Meskauskas, A., Dinman. J.D., and Simon, A.E.  (2011).  Ribosome binding to a 5’ translational enhancer is altered in the presence of the 3’UTR in cap-independent translation of Turnip crinkle virus.  J. Virol. 85, 4638-4653.

Yuan, X., Shi, K., Young, M. Y. L., and Simon, A. E. (2010).  The terminal loop of a 3’ proximal hairpin plays a critical role in the structure of the 3’ region of Turnip crinkle virus and the RdRp-mediated conformational switch.  Virology 402, 271-280.

Zuo, X, Wang, J., Yu, P.,  Eyler, D., Xu, H.,  Starich, M.R., Tiede, D.M., Simon, A.E., Kasprzak, W., Schwieters, C.D., Shapiro, B.A.,  and Wang, Y.-W.  (2010) The cap-independent translational enhancer and ribosome binding structure element in 3' UTR of Turnip crinkle virus RNA folds into a tRNA-like shape in solution.  Proc. Natl. Acad. Sci. USA  107, 1385-1390.

Simon, A. E., and Gerhke, L.  (2009)  Regulation of virus gene Expression by RNA conformational switches.  BBA 1789, 571-583.

Yuan, X., Shi, K., Meskauskas, A. and Simon, A.E.  (2009)  The 3’ End of Turnip crinkle virus contains a highly interactive structure with a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase.  RNA 15, 1849-1864.

Guo, R., Lin, W., Zhang, J., Simon, A. E., and Kushner, D. B.  (2009).  Structural plasticity and rapid evolution in a viral RNA revealed by in vivo genetic selection.  J. Virol. 83, 927-939.

McCormack, J. C., Yuan, X., Yingling, Y. G., Zamora, R. E., Shapiro, B. A., and Simon, A. E.  (2008).  Structural domains within the 3' UTR of Turnip crinkle virus.  J. Virol. 82, 8706-8720.

Stupina, V. A., Meskauskas, A., McCormack, J. C., Yingling, Y. G., Kasprzak, W., Shapiro, B. A., Dinman, J. D., and Simon, A. E.  (2008).  The 3' proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits.  RNA 14, 2379-2393.

Zhang, J., Zhang, G., Guo, R., Shapiro, B. and Simon, A. E. (2006)  A pseudoknot in a pre-active form of a viral RNA is part of a structural switch activating minus-strand synthesis.  J. Virol 80, 9181-9191.

Zhang, J., Zhang,  G., McCormack, J. and Simon, A. E. (2006).  Evolution of virus-derived sequences for high level replication of a subviral RNA.  Virology 351, 476-488.

Sun, X., and Simon, A. E.  (2006) A cis-replication element functions in both orientations to enhance replication of Turnip crinkle virus.  Virology 352, 39-51.

Zhang, G., Zhang, J., George, A. T., Baumstark, T., and Simon, A. E.  (2006)  Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA.  RNA 12, 147-162.

Zhang, J. and Simon, A. E.  (2005)  Importance of sequence and structural elements within  A viral replication repressor.  Virology 333, 301-315.,

Sun, X., Zhang, G., and Simon, A. E. (2005)  Short internal sequences involved in RNA replication and virion accumulation in a subviral RNA of Turnip crinkle virus. J. Virol. 79, 512-524.

Zhang, J., Stuntz, R. M., and Simon, A. E.  (2004) Analysis of a viral replication repressor:  Sequence requirements in the large symmetrical loop. Virology 326,90-102. 

Zhang G, Zhang J, and Simon AE. (2004).  Repression and derepression of minus-strand synthesis in a plus-strand RNA virus replicon.  J. Virol. 78, 7619-7633.

McCormack, J. and Simon, A. E. (2004)  Biased hypermutagensis associated with mutations in an untranslated hairpin of an RNA virus.  J. Virol. 78, 7813-7817.

Sun, X., and Simon, A.E (2003) Fitness of a Turnip crinkle virus satellite RNA correlates with a sequence-nonspecific hairpin and flanking sequences that enhance replication and repress accumulation of virions.  J. Virol. 77, 7880-7889.

Zhang, G. and Simon, A. E. (2003)  A multifunctional turnip crinkle virus replication enhancer revealed by in vivo functional selex.  J. Mol. Biol. 326, 35-48.

Guan, H., Carpenter, C. D., and Simon, A. E.  (2000)  Requirement of a 5’-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with TCV. Virology 268, 355-363.

Guan, H and Simon, A. E.   2000.  Polymerization of non-template bases prior to transcription initiation by an RNA-dependent RNA polymerase:  A novel activity involved in 3’-end repair of viral RNAs.  Proc. Natl. Acad. Sci. 97, 12451-12456.

Guan, H., Carpenter, C. D., and Simon, A. E.  (2000)  Analysis of cis-acting sequences involved in plus-strand synthesis of a TCV-associated satellite RNA identifies a new carmovirus replication element.  Virology 268, 345-354.

Wang, J.L., and Simon, A.E.  (2000)  3'-end stem-loops of the subviral RNAs associated with turnip crinkle virus are involved in symptom modulation and coat protein binding.  J Virol 74, 6528-6537.

Wang, J., Carpenter, C.D. and Simon, A. E. (1999) Minimal Sequence and Structural Requirements of A Subgenomic RNA Promoter for Turnip Crinkle Virus Virology 253, 327-336

Carpenter, C.D. and Simon, A. E. (1998) Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection Nucleic Acids Research, 1998, Vol. 26, 2426 - 2432

Nagy, P. D., Carpenter, C. D.,  and Simon, A. E.   1997.  A novel 3' end repair mechanism in an RNA virus.  Proc. Natl. Acad. Sci. USA. 94, 1113-1118.  PMID: 9037015

Guan, H., Song, C., and Simon, A. E. (1997) RNA promoters located on (-)-strands of a subviral RNA associated with turnip crinkle virus RNA 3:1401-1412

Stupina, V., and Simon, A. E. (1997) Analysis in vivo of trunip crinkle virus satellite RNA C variants with mutations in the 3' terminal minus strand promoter. Virology, 238, 470-477

Wang, J. and Simon, A. E. (1997) Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro. Virology 232, 174-186.

Song, C. and Simon, A. E. (1995) Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase. J. Mol. Biol. 254, 6-14

Song, C., and Simon, A. E. (1995) Synthesis of novel products in vitro by an RNA-dependent RNA polymerase. J. Virol. 69. 4020-4028.

Carpenter, C.D., Oh, J.-W., Zhang, C., and Simon, A. E. (1995) Involvement of a stem-loop structure in the location of junction sites in viral RNA recombination. J. Mol. Biol. 245, 608-622.

Zhang, C., and Simon, A. E. (1994) Effect of template size on replication of defective interfering RNAs. J. Virol. 68, 8466-8469.

Song, C., and Simon, A.E. (1994) RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+)- and (-)-strands of virus-associated RNAs. Proc. Natl. Acad Sci.USA. 91, 8792-8796.