Cis-acting Elements in the 3' Region of Potato virus X are Required for HostProtein Binding

권선정,김국형,Cynthia Hemenway 한국식물병리학회 2006 Plant Pathology Journal Vol.22 No.2

The 3' region of Potato virus X (PVX) has the 74 nt 3'- nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3' NTR were previously demonstrated as important cis-acting elements. To investigate the binding of these cis-acting elements within 3' end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3' end of PVX RNA showed that the 3' end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (Δ10, SL3B, SL2A and Δ21; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cisacting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3' end which are important for minus and plus-strand RNA accumulation are also required for host protein binding.

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

박미리,박상호,조상윤,Cynthia L. Hemenway,최홍수,손성한,김국형 한국식물병리학회 2008 Plant Pathology Journal Vol.24 No.3

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 basepairing 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 (Δ12) 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 (Δ8) 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.

Cis-acting Elements in the 3' Region of Potato virus X are Required for Host Protein Binding

Kwon, Sun-Jung,Kim, Kook-Hyung,Hemenway Cynthia The Korean Society of Plant Pathology 2006 Plant Pathology Journal Vol.22 No.2

The 3' region of Potato virus X (PVX) has the 74 nt 3'-nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3' NTR were previously demonstrated as important cis-acting elements. To Investigate the binding of these cis-acting elements within 3' end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3' end of PVX RNA showed that the 3' end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (${\Delta}10$10, SL3B, SL2A and ${\Delta}21$; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cis-acting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3' end which are important for minus and plus-strand RNA accumulation are also required for host protein binding.

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

Park, Mi-Ri,Park, Sang-Ho,Cho, Sang-Yun,Hemenway, Cynthia L.,Choi, Hong-Soo,Sohn, Seong-Han,Kim, Kook-Hyung The Korean Society of Plant Pathology 2008 Plant Pathology Journal Vol.24 No.3

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(${\Delta}12$) 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(${\Delta}8$) 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.

<i>cis</i>-Acting sequences required for coat protein binding and in vitro assembly of <i>Potato virus X</i>

Kwon, Sun-Jung,Park, Mi-Ri,Kim, Ki-Woo,Plante, Carol A.,Hemenway, Cynthia L.,Kim, Kook-Hyung Elsevier 2005 Virology Vol.334 No.1

<P><B>Abstract</B></P><P>The 5′ region of <I>Potato virus X</I> (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.</P>