@article{bamunusinghe_hemenway_nelson_sanderfoot_ye_silva_payton_verchot-lubicz_2009, title={Analysis of potato virus X replicase and TGBp3 subcellular locations}, volume={393}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2009.08.002}, abstractNote={Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.}, number={2}, journal={VIROLOGY}, author={Bamunusinghe, Devinka and Hemenway, Cynthia L. and Nelson, Richard. S. and Sanderfoot, Anton A. and Ye, Chang M. and Silva, Muniwarage A. T. and Payton, M. and Verchot-Lubicz, Jeanmarie}, year={2009}, month={Oct}, pages={272–285} }
 @article{park_kwon_choi_hemenway_kim_2008, title={Mutations that alter a repeated ACCA element located at the 5 ' end of the Potato virus X genome affect RNA accumulation}, volume={378}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2008.05.004}, abstractNote={The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.}, number={1}, journal={VIROLOGY}, author={Park, Mi-Ri and Kwon, Sun-Jung and Choi, Hong-Soo and Hemenway, Cynthia L. and Kim, Kook-Hyung}, year={2008}, month={Aug}, pages={133–141} }
 @article{park_park_cho_hemenway_choi_sohn_kim_2008, title={RNA-RNA interactions between RNA elements at the 5 ' end and at the upstream of sgRNA of RNA genome are required for Potato virus X RNA replication}, volume={24}, ISSN={["2093-9280"]}, DOI={10.5423/PPJ.2008.24.3.289}, abstractNote={RNA-RNA interactions and the dynamic RNA conformations are important regulators in virus replication in several RNA virus systems and may also involved in the regulation of many important virus life cycle phases, including translation, replication, assembly, and switches in these important stages. The 5` non-translated region of Potato virus X(PVX) contains multiple cis-acting elements that facilitate various viral processes. It has previously been proposed that RNA-RNA interactions between various RNA elements present in PVX RNA genome are required for PVX RNA accumulation(Hu et al., 2007; Kim and Hemenway, 1999). This model was based on the potential base-pairing between conserved sequence elements at the upstream of subgenomic RNAs(sgRNAs) and at the 5` and 3` end of RNA genome. We now provide more evidence that RNA-RNA base-pairing between elements present at the 5` end and upstream of each sgRNA is required for efficient replication of genomic and subgenomic plus-strand RNA accumulation. Site-directed mutations introduced at the 5` end of plus-strand RNA replication defective mutant() increasing base-pairing possibility with conserved sequence elements located upstream of each sgRNAs restored genomic and subgenomic plus-strand RNA accumulation and caused symptom development in inoculated Nicotiana benthamiana plants. Serial passage of a deletion mutant() caused more severe symptoms and restored wild type sequences and thus retained possible RNA-RNA base-pairing. Altogether, these results indicate that the RNA element located at the 5` end of PVX genome involved in RNA-RNA interactions and play a key role in high-level accumulation of plus-strand RNA in vivo.}, number={3}, journal={PLANT PATHOLOGY JOURNAL}, author={Park, Mi-Ri and Park, Sang-Ho and Cho, Sang-Yun and Hemenway, Cynthia L. and Choi, Hong-Soo and Sohn, Seong Han and Kim, Kook-Hyung}, year={2008}, month={Sep}, pages={289–295} }
 @article{hu_pillai-nair_hemenway_2007, title={Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis}, volume={13}, ISSN={["1469-9001"]}, DOI={10.1261/rna.243607}, abstractNote={Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5′ terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3′ NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3′ terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3′ NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3′ hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.}, number={2}, journal={RNA}, author={Hu, Bin and Pillai-Nair, Neeta and Hemenway, Cynthia}, year={2007}, month={Feb}, pages={267–280} }
 @article{kwon_park_kim_plante_hemenway_kim_2005, title={cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X}, volume={334}, ISSN={["1089-862X"]}, DOI={10.1016/j.virol.2005.01.018}, abstractNote={The 5′ region of Potato virus X (PVX) RNA containing an AC-rich single-stranded region and stem–loop 1 (SL1) has been shown to be important for PVX replication (Miller, E.D., Plante, C.A., Kim, K.-H., Brown, J.W., Hemenway, C., 1998. Stem–loop structure in the 5′ region of potato virus X genome required for plus-strand RNA accumulation. J. Mol. Biol. 284, 591–608.). Here, we describe the involvement of SL1 for binding to the PVX coat protein (CP) using an in vitro assembly system and various deletion mutants of the 5′ region of PVX RNA. Internal and 5′ terminal deletions of the 5′-nontranslated region of PVX RNA were assessed for their effects on formation of assembled virus-like particles (VLPs). Mutant RNAs that contain the top region of SL1 or sequences therein bound to CP to form VLPs. In contrast, transcripts of mutants that disrupt SL1 RNA structure were unable to form VLPs. SELEX was used to further confirm the specific RNA recognition of PVX CP using RNA transcripts containing randomized sequences of the upper portion of SL1. Wild-type (wt) sequences along with many other sequences that resemble SL1 structure were selected after fourth and fifth rounds of SELEX (27.0% and 44.4%, respectively). RNA transcripts from several SELEX winners that are predicted to form stable stem–loop structures very closely resembling wt PVX SL1 VLPs. RNA transcripts not predicted to form secondary structures similar to SL1 did not form VLPs in vitro. Taken together, our results suggest that RNA secondary structural elements within SL1 and/or sequences therein are crucial for formation of VLPs and are required for the specific recognition by the CP subunit.}, number={1}, journal={VIROLOGY}, author={Kwon, SJ and Park, MR and Kim, KW and Plante, CA and Hemenway, CL and Kim, KH}, year={2005}, month={Mar}, pages={83–97} }
 @article{pillai-nair_kim_hemenway_2003, title={Cis-acting regulatory elements in the potato virus X 3 ' non-translated region differentially affect minus-strand and plus-strand RNA accumulation}, volume={326}, ISSN={["0022-2836"]}, DOI={10.1016/S0022-2836(02)01369-4}, abstractNote={The 72 nt 3′ non-translated region (NTR) of potato virus X (PVX) RNA is identical in all sequenced PVX strains and contains sequences that are conserved among all potexviruses. Computer folding of the 3′ NTR sequence predicted three stem-loop structures (SL1, SL2, and SL3 in the 3′ to 5′ direction), which generally were supported by solution structure analyses. The importance of these sequence and/or structural elements to PVX RNA accumulation was further analyzed by inoculation of Nicotiana tabacum (NT-1) protoplasts with PVX transcripts containing mutations in the 3′ NTR. Analyses of RNA accumulation by S1 nuclease protection indicated that multiple sequence elements throughout the 3′ NTR were important for minus-strand RNA accumulation. Formation of SL3 was required for accumulation of minus-strand RNA, whereas SL1 and SL2 formation were less important. However, sequences within all of these predicted structures were required for minus-strand RNA accumulation, including a conserved hexanucleotide sequence element in the loop of SL3, and the CU nucleotide in a U-rich sequence within SL2. In contrast, 13 nucleotides that were predicted to reside in SL1 could be deleted without any significant reduction in minus or plus-strand RNA levels. Potential polyadenylation signals (near upstream elements; NUEs) in the 3′ NTR of PVX RNA were more important for plus-strand RNA accumulation than for minus-strand RNA accumulation. In addition, one of these NUEs overlapped with other sequence required for optimal minus-strand RNA levels. These data indicate that the PVX 3′ NTR contains multiple, overlapping elements that influence accumulation of both minus and plus-strand RNA.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Pillai-Nair, N and Kim, KH and Hemenway, C}, year={2003}, month={Feb}, pages={701–720} }
 @misc{batten_yoshinari_hemenway_2003, title={Potato virus X: a model system for virus replication, movement and gene expression}, volume={4}, ISSN={["1364-3703"]}, DOI={10.1046/j.1364-3703.2003.00156.x}, abstractNote={SUMMARY}, number={2}, journal={MOLECULAR PLANT PATHOLOGY}, author={Batten, JS and Yoshinari, S and Hemenway, C}, year={2003}, month={Mar}, pages={125–131} }
 @article{kim_kwon_hemenway_2002, title={Cellular protein binds to sequences near the 5 ' terminus of potato virus X RNA that are important for virus replication}, volume={301}, ISSN={["0042-6822"]}, DOI={10.1006/viro.2002.1559}, abstractNote={The sequences in the 5'-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5'-NTR. These analyses showed that the 5' region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1-46 at the 5' terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10-13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5' terminus of the viral genomic RNA.}, number={2}, journal={VIROLOGY}, author={Kim, KH and Kwon, SJ and Hemenway, C}, year={2002}, month={Sep}, pages={305–312} }
 @article{osman_hemenway_buck_2000, title={Role of the 3 ' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase in vitro}, volume={74}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.74.24.11671-11680.2000}, abstractNote={ABSTRACT}, number={24}, journal={JOURNAL OF VIROLOGY}, author={Osman, TAM and Hemenway, CL and Buck, KW}, year={2000}, month={Dec}, pages={11671–11680} }
 @article{plante_kim_pillai-nair_osman_buck_hemenway_2000, title={Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates}, volume={275}, ISSN={["0042-6822"]}, DOI={10.1006/viro.2000.0512}, abstractNote={We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.}, number={2}, journal={VIROLOGY}, author={Plante, CA and Kim, KH and Pillai-Nair, N and Osman, TAM and Buck, KW and Hemenway, CL}, year={2000}, month={Sep}, pages={444–451} }
 @article{kim_hemenway_1999, title={Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation}, volume={5}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838299982006}, abstractNote={Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.}, number={5}, journal={RNA}, author={Kim, KH and Hemenway, CL}, year={1999}, month={May}, pages={636–645} }
 @article{miller_kim_hemenway_1999, title={Restoration of a stem-loop structure required for potato virus X RNA accumulation indicates selection for a mismatch and a GNRA tetraloop}, volume={260}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1999.9843}, abstractNote={The 5' region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions onto Nicotiana benthamiana plants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55-C78 mismatch to a G-C pair, relocation of this mismatch within the central region of SC, or deletion of C55-C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55-C78 mismatch to an A-C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C-C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure.}, number={2}, journal={VIROLOGY}, author={Miller, ED and Kim, KH and Hemenway, C}, year={1999}, month={Aug}, pages={342–353} }
 @inbook{miller_hemenway_1998, title={History of coat protein-mediated protection}, volume={81}, DOI={10.1385/0-89603-385-6:25}, abstractNote={Since the first demonstration by Powell-Abel et al. (1) that plants engineered to express the tobacco mosaic virus (TMV) coat protein (CP) gene can resist corresponding viral infection, a decade of research on CP-mediated protection (CPMP) has produced transgenic plants resistant to a multitude of different plant viruses. This field rapidly progressed from testing resistance in model plant systems under growth chamber conditions to conducting field trials on agronomically significant crops such as tomato, potato, sugarbeets, melons, cucumber, tobacco, and rice. In addition, this approach to protection has been extended by expression of other viral sequences corresponding to satellite RNAs, antisense transcripts, sense transcripts, defective interfering sequences, nonstructural genes, portions of genes, and mutated genes in transgenic plants.}, booktitle={Plant virology protocols: from virus isolation to transgenic resistance / edited by Gary D. Foster and Sally C. Taylor. (Methods in molecular biology (Clifton, N.J.) ; 81)}, publisher={Totowa, N.J.: Humana Press}, author={Miller, E. D. and Hemenway, C.}, year={1998}, pages={25–38} }
 @article{miller_plante_kim_brown_hemenway_1998, title={Stem-loop structure in the 5 ' region of potato virus X genome required for plus-strand RNA accumulation}, volume={284}, ISSN={["1089-8638"]}, DOI={10.1006/jmbi.1998.2174}, abstractNote={Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem-loop 2 (SL2; nt 143-183), within the 5' 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast system. In general, mutations that reduced genomic plus-strand RNA accumulation similarly affected coat protein accumulation, indicating that subgenomic plus-strand RNA was also affected. In contrast, minus-strand RNA levels remained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as well as specific nucleotides within this loop, were also required. Several modifications were made to the terminal GAAA tetraloop, a motif known for enhanced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stabilization of the structure. These data indicate that multiple features of SL1 are critical for accumulation of PVX plus-strand RNA.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Miller, ED and Plante, CA and Kim, KH and Brown, JW and Hemenway, C}, year={1998}, month={Dec}, pages={591–608} }
 @article{kim_hemenway_1997, title={Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation}, volume={232}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1997.8565}, abstractNote={The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.}, number={1}, journal={VIROLOGY}, author={Kim, KH and Hemenway, C}, year={1997}, month={May}, pages={187–197} }