@article{park_sit_kim_lommel_2013, title={The red clover necrotic mosaic virus capsid protein N-terminal amino acids possess specific RNA binding activity and are required for stable virion assembly}, volume={176}, ISSN={["0168-1702"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84881374230&partnerID=MN8TOARS}, DOI={10.1016/j.virusres.2013.05.014}, abstractNote={The red clover necrotic mosaic virus (RCNMV) bipartite RNA genome is packaged into two virion populations containing either RNA-1 and RNA-2 or multiple copies of RNA-2 only. To understand this distinctive packaging scheme, we investigated the RNA-binding properties of the RCNMV capsid protein (CP). Maltose binding protein-CP fusions exhibited the highest binding affinities for RNA probes containing the RNA-2 trans-activator or the 3′ non-coding region from RNA-1. Other viral and non-viral RNA probes displayed CP binding but to a much lower degree. Deletion of the highly basic N-terminal 50 residues abolished CP binding to viral RNA transcripts. In planta studies of select CP deletion mutants within this N-terminal region revealed that it was indispensable for stable virion formation and the region spanning CP residues 5–15 is required for systemic movement. Thus, the N-terminal region of the CP is involved in both producing two virion populations due to its RNA binding properties and virion stability.}, number={1-2}, journal={VIRUS RESEARCH}, publisher={Elsevier BV}, author={Park, Sang-Ho and Sit, Tim L. and Kim, Kook-Hyung and Lommel, Steven A.}, year={2013}, month={Sep}, pages={107–118} }
 @article{park_sit_kim_lommel_2012, title={The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation}, volume={13}, ISSN={["1364-3703"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84864388918&partnerID=MN8TOARS}, DOI={10.1111/j.1364-3703.2011.00784.x}, abstractNote={SUMMARY}, number={7}, journal={MOLECULAR PLANT PATHOLOGY}, publisher={Wiley}, author={Park, Sang-Ho and Sit, Tim L. and Kim, Kook-Hyung and Lommel, Steven A.}, year={2012}, month={Sep}, pages={744–754} }
 @article{powers_sit_qu_morris_kim_lommel_2008, title={A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement}, volume={21}, ISSN={["1943-7706"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-47549110190&partnerID=MN8TOARS}, DOI={10.1094/MPMI-21-7-0879}, abstractNote={ The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVΔ92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. }, number={7}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, publisher={Scientific Societies}, author={Powers, Jason G. and Sit, Tim L. and Qu, Feng and Morris, T. Jack and Kim, Kook-Hyung and Lommel, Steven A.}, year={2008}, month={Jul}, pages={879–890} }
 @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{powers_sit_heinsohn_george_kim_lommel_2008, title={The Red clover necrotic mosaic virus RNA-2 encoded movement protein is a second suppressor of RNA silencing}, volume={381}, ISSN={["0042-6822"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-54549091972&partnerID=MN8TOARS}, DOI={10.1016/j.virol.2008.09.004}, abstractNote={The replication complex of Red clover necrotic mosaic virus (RCNMV) has been shown to possess silencing suppression activity. Here a newly developed viral-based assay for the identification of silencing suppression activity was used to provide evidence for a second, mechanistically distinct method of silencing suppression provided for by the RCNMV movement protein (MP). This new assay relies on Turnip crinkle virus with its capsid protein replaced with green fluorescent protein to act as a reporter (TCV-sGFP). In the presence of a protein with silencing suppression activity TCV-sGFP readily moves from cell-to-cell, but in the absence of such a protein TCV-sGFP is confined to small foci of infection. This TCV-sGFP assay was used to identify MP as a suppressor of RNA silencing, to delimit essential amino acids for this activity and uncouple silencing and movement functions.}, number={2}, journal={VIROLOGY}, publisher={Elsevier BV}, author={Powers, Jason G. and Sit, Tim L. and Heinsohn, Curtis and George, Carol G. and Kim, Kook-Hyung and Lommel, Steven A.}, year={2008}, month={Nov}, pages={277–286} }
 @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} }
 @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{kim_1999, title={Recent development in detection and identification of fruit tree viruses}, volume={15}, number={4}, journal={Plant Pathology Journal}, author={Kim, K.-H.}, year={1999}, pages={199} }
 @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} }
 @article{kim_lommel_1998, title={Sequence element required for efficient -1 ribosomal frameshifting in red clover necrotic mosaic dianthovirus}, volume={250}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1998.9358}, abstractNote={The RNA-1 of the bipartite red clover necrotic mosaic dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5' proximal and internal open reading frames by a -1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A beta-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient -1 ribosomal frameshift expression in vivo. A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA-1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiency in vitro or infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshifting in vitro and biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.}, number={1}, journal={VIROLOGY}, author={Kim, KH and Lommel, SA}, year={1998}, month={Oct}, pages={50–59} }
 @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} }
 @article{kim_hemenway_1996, title={The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis}, volume={70}, number={8}, journal={Journal of Virology}, author={Kim, K.-H. and Hemenway, C.}, year={1996}, pages={5533} }