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Alanine scanning of poliovirus 2CATPase reveals new genetic evidence that capsid protein/2CATPase interactions are essential for morphogenesis. J Virol.2012; 86: 9964–9975. doi
"... Polypeptide 2CATPase is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In ..."
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Polypeptide 2CATPase is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate con-ditional-lethal mutations in hitherto-uncharacterized domains of the 2CATPase polypeptide, particularly those involved in mor-phogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2CATPase coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K6A/ K7A) that produced a suppressor mutation (G1R) and a novel 2B^2C ATPase cleavage site (Q^R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2CATPase polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2CATPase that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2CATPase. Most importantly, the identification of suppres-
Email correspondence
- Danny Lee, OK Farm Service Agency and Victoria Thompson, ICF International. July
, 2007
"... Insulin autoimmune syndrome induced by methimazole in a Korean girl with Graves ’ disease Case report Hypoglycemia was detected in a 15-year-old girl due to loss of consciousness. She was diagnosed with Graves ’ disease and was being treated with methimazole for the past 4 months. A paradoxically in ..."
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Insulin autoimmune syndrome induced by methimazole in a Korean girl with Graves ’ disease Case report Hypoglycemia was detected in a 15-year-old girl due to loss of consciousness. She was diagnosed with Graves ’ disease and was being treated with methimazole for the past 4 months. A paradoxically increased insulin levels was found when she suffered from the hypoglycemic episode. An imaging study showed no mass lesion in the pancreas, and insulin antibodies were found in the serum. She was diagnosed with insulin autoimmune syndrome. Her HLA typing was performed, and it revealed HLA-DRB1 *04:06. The patient was treated with a corticosteroid for 2 months. After discontinuing the steroid, the insulin antibody titer decreased dramatically, and she did not have any episode of hypoglycemia since. This is the first report of insulin autoimmune syndrome in a Korean girl, and we have revealed the connection between HLA type and insulin autoimmune syndrome in Korea.
Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags
, 2009
"... The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease ..."
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The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indis-tinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.
Open Access
"... Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost ..."
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Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis. Background Enterovirus type 71 (EV71) is the causative agent of several
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, 2010
"... Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, ..."
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Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF,
C Virus Replication �
, 2007
"... These include: This article cites 55 articles, 28 of which can be accessed free at: ..."
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These include: This article cites 55 articles, 28 of which can be accessed free at:
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"... Running title: Novel heterocyclic compounds inhibiting HCV replication Abstract: 222 words Text: 6795 words ..."
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Running title: Novel heterocyclic compounds inhibiting HCV replication Abstract: 222 words Text: 6795 words
REFERENCES CONTENT ALERTS
, 1997
"... These include: This article cites 44 articles, 16 of which can be accessed free at: ..."
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These include: This article cites 44 articles, 16 of which can be accessed free at:
REFERENCES CONTENT ALERTS
, 2007
"... These include: This article cites 41 articles, 27 of which can be accessed free at: ..."
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These include: This article cites 41 articles, 27 of which can be accessed free at:
