The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform
Elizabeth Rieder - One of the best experts on this subject based on the ideXlab platform.
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Bioinformatics and Molecular Analysis of the Evolutionary Relationship between Bovine Rhinitis A Viruses and Foot-And-Mouth Disease Virus.
Bioinformatics and biology insights, 2016Co-Authors: Devendra K. Rai, Paul Lawrence, Steve J. Pauszek, M. E. Piccone, Nick J. Knowles, Elizabeth RiederAbstract:Bovine rhinitis viruses (BRVs) cause mild respiratory disease of cattle. In this study, a near full-length genome sequence of a virus named RS3X (formerly classified as bovine rhinovirus type 1), isolated from infected cattle from the UK in the 1960s, was obtained and analyzed. Compared to other closely related Aphthoviruses, major differences were detected in the leader protease (Lᵖʳᵒ), P1, 2B, and 3A proteins. Phylogenetic analysis revealed that RS3X was a member of the species bovine rhinitis A virus (BRAV). Using different codon-based and branch-site selection models for Aphthoviruses, including BRAV RS3X and foot-and-mouth disease virus, we observed no clear evidence for genomic regions undergoing positive selection. However, within each of the BRV species, multiple sites under positive selection were detected. The results also suggest that the probability (determined by Recombination Detection Program) for recombination events between BRVs and other Aphthoviruses, including foot-and-mouth disease virus was not significant. In contrast, within BRVs, the probability of recombination increases. The data reported here provide genetic information to assist in the identification of diagnostic signatures and research tools for BRAV.
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Characterization of a chimeric foot-and-mouth disease virus bearing a bovine rhinitis B virus leader proteinase
Virology, 2013Co-Authors: Sabena Uddowla, Devendra K. Rai, Juan M. Pacheco, Christopher R. Larson, Elizabeth Bishop, Luis L. Rodriguez, Jonathan Arzt, Elizabeth RiederAbstract:Abstract Bovine rhinitis B virus (BRBV) shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). This study examined if the BRBV leader proteinase (L pro ) could functionally replace that of FMDV. A mutant A 24 L BRV 3D YR FMDV engineered with the BRBV L pro and an antigenic marker in the 3D polymerase exhibited growth properties and eIF4G cleavage similar to parental A 24 WT virus. The A 24 L BRV 3D YR type I interferon activity in infected bovine cells resembled that of A 24 LL virus that lacks L pro , but this effect was less pronounced for A 24 L BRV 3D YR infected porcine cells. In vivo studies showed that the A 24 L BRV 3D YR virus was attenuated in cattle, and exhibited low virulence in pigs exposed by direct contact. The mutant virus induced protective immunity in cattle against challenge with parental A 24 WT. These results provide evidence that L pro of different Aphthoviruses are not fully functionally interchangeable and have roles that may depend on the nature of the infected host.
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Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).
International Journal of Molecular Sciences, 2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2A resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.
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Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA
2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Abstract: Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43 % identical polyprotein sequence and as much as 67 % identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D pol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D pol using a combination of different computational tools. Model structures of BRBV 3D pol were verified for their stereochemical quality and accuracy. The BRBV 3D pol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP
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Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)
MDPI AG, 2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D<sup>pol</sup>). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D<sup>pol</sup> using a combination of different computational tools. Model structures of BRBV 3D<sup>pol</sup> were verified for their stereochemical quality and accuracy. The BRBV 3D<sup>pol</sup> structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D<sup>pol</sup> (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D<sup>pol</sup> as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D<sup>pols</sup> is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D<sup>pol</sup> compared to FMDV 3D<sup>pol</sup>, indicate that despite a very high homology to FMDV 3D<sup>pol</sup>, BRBV 3D<sup>pol</sup> may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to <em>Aphthovirus</em> RdRps
Devendra K. Rai - One of the best experts on this subject based on the ideXlab platform.
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Bioinformatics and Molecular Analysis of the Evolutionary Relationship between Bovine Rhinitis A Viruses and Foot-And-Mouth Disease Virus.
Bioinformatics and biology insights, 2016Co-Authors: Devendra K. Rai, Paul Lawrence, Steve J. Pauszek, M. E. Piccone, Nick J. Knowles, Elizabeth RiederAbstract:Bovine rhinitis viruses (BRVs) cause mild respiratory disease of cattle. In this study, a near full-length genome sequence of a virus named RS3X (formerly classified as bovine rhinovirus type 1), isolated from infected cattle from the UK in the 1960s, was obtained and analyzed. Compared to other closely related Aphthoviruses, major differences were detected in the leader protease (Lᵖʳᵒ), P1, 2B, and 3A proteins. Phylogenetic analysis revealed that RS3X was a member of the species bovine rhinitis A virus (BRAV). Using different codon-based and branch-site selection models for Aphthoviruses, including BRAV RS3X and foot-and-mouth disease virus, we observed no clear evidence for genomic regions undergoing positive selection. However, within each of the BRV species, multiple sites under positive selection were detected. The results also suggest that the probability (determined by Recombination Detection Program) for recombination events between BRVs and other Aphthoviruses, including foot-and-mouth disease virus was not significant. In contrast, within BRVs, the probability of recombination increases. The data reported here provide genetic information to assist in the identification of diagnostic signatures and research tools for BRAV.
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Characterization of a chimeric foot-and-mouth disease virus bearing a bovine rhinitis B virus leader proteinase
Virology, 2013Co-Authors: Sabena Uddowla, Devendra K. Rai, Juan M. Pacheco, Christopher R. Larson, Elizabeth Bishop, Luis L. Rodriguez, Jonathan Arzt, Elizabeth RiederAbstract:Abstract Bovine rhinitis B virus (BRBV) shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). This study examined if the BRBV leader proteinase (L pro ) could functionally replace that of FMDV. A mutant A 24 L BRV 3D YR FMDV engineered with the BRBV L pro and an antigenic marker in the 3D polymerase exhibited growth properties and eIF4G cleavage similar to parental A 24 WT virus. The A 24 L BRV 3D YR type I interferon activity in infected bovine cells resembled that of A 24 LL virus that lacks L pro , but this effect was less pronounced for A 24 L BRV 3D YR infected porcine cells. In vivo studies showed that the A 24 L BRV 3D YR virus was attenuated in cattle, and exhibited low virulence in pigs exposed by direct contact. The mutant virus induced protective immunity in cattle against challenge with parental A 24 WT. These results provide evidence that L pro of different Aphthoviruses are not fully functionally interchangeable and have roles that may depend on the nature of the infected host.
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Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).
International Journal of Molecular Sciences, 2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2A resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.
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Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA
2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Abstract: Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43 % identical polyprotein sequence and as much as 67 % identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D pol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D pol using a combination of different computational tools. Model structures of BRBV 3D pol were verified for their stereochemical quality and accuracy. The BRBV 3D pol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP
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Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)
MDPI AG, 2012Co-Authors: Devendra K. Rai, Elizabeth RiederAbstract:Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the Aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D<sup>pol</sup>). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D<sup>pol</sup> using a combination of different computational tools. Model structures of BRBV 3D<sup>pol</sup> were verified for their stereochemical quality and accuracy. The BRBV 3D<sup>pol</sup> structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D<sup>pol</sup> (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D<sup>pol</sup> as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D<sup>pols</sup> is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D<sup>pol</sup> compared to FMDV 3D<sup>pol</sup>, indicate that despite a very high homology to FMDV 3D<sup>pol</sup>, BRBV 3D<sup>pol</sup> may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to <em>Aphthovirus</em> RdRps
Stanley M. Lemon - One of the best experts on this subject based on the ideXlab platform.
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Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14
Journal of Virology, 2002Co-Authors: Yan Yang, Eckard Wimmer, Rene Rijnbrand, Kevin L. Mcknight, Aniko V. Paul, Annette Martin, Stanley M. LemonAbstract:The family Picornaviridae is one of the largest groups of nonenveloped, single-stranded, positive-sense RNA viruses. It comprises nine distinct genera, of which five (the enteroviruses, rhinoviruses, Aphthoviruses, cardioviruses, and hepatoviruses) are important human and animal pathogens (23). These genera are distinguished from each other by differences in the proteins they express, but the RNA genomes of each have a similar organization, with a lengthy 5′ nontranslated region (5′ NTR) followed by a single, large open reading frame encoding a single polyprotein, a 3′ NTR, and a genetically templated 3′-terminal poly(A) tail (28). Since it is positive sense, the virion RNA acts directly as message, programming the translation of the polyprotein following its release into the cytoplasm. Virion RNA and synthetic genome-length RNA derived from recombinant cDNA clones are thus infectious when transfected into permissive cells, giving rise to virus particles and subsequent rounds of virus replication (22). An unusual feature of the genomic RNA of picornaviruses is that it lacks the 5′-terminal cap structure present in most eucaryotic mRNAs and is instead covalently linked to a small, virally encoded protein, VPg (3, 8). An internal ribosomal entry site located within the 5′ NTR directs the cap-independent translation of the polyprotein (6, 19), so that the cellular translational machinery thus bypasses the 5′ end of the genome. The polyprotein contains three major functional segments, defined in part by the order of cleavage events that occur during its processing by one or more viral proteases (26). In the case of the enteroviruses and rhinoviruses, the most N-terminal segment, P1, contains four capsid proteins, VP4, VP2, VP3, and VP1, while the P2 and P3 segments are comprised of nonstructural proteins involved in protein maturation and RNA replication. These include 2Apro, 2B, 2C, 3A, 3B (VPg), 3Cpro, and 3Dpol and their functional precursors, 2BC, 3AB, and 3CDpro (15). In broad outline, the replication of picornaviral RNAs follows a two-step process. Upon entry into host cells and uncoating of the virus, the incoming positive-strand genome is transcribed into complementary minus-strand RNA by a replicase complex, the catalytic unit of which, 3Dpol, uses a uridylylated form of VPg (VPg-pUpU) as a primer (18). This minus-strand RNA then serves as a template for the production of new, plus-strand, progeny genomes. Although the basic steps of replication are well known, relatively little is understood about the details of these processes. One of the important, yet incompletely answered questions is how the viral replicase specifically selects only viral RNA species for amplification in these reactions, because the 3′-terminal poly(A) sequence of the genomic RNA is indistinguishable from the 3′ termini of cellular mRNAs. Until recently, it was generally believed that the 5′ and 3′ NTRs of picornaviral RNA contain the cis-acting signals necessary for the initiation of viral RNA replication (28). The specificity of genome amplification was thought to result from interactions of the replicase complex with unique cis-acting signals located in the 5′ and 3′ NTRs (1, 13, 14, 21, 22, 25, 27). However, McKnight and Lemon (11, 12) demonstrated that an internal cis-acting replication element (cre) was necessary for the initiation of RNA synthesis during replication of human rhinovirus type 14 (HRV-14). Although this replication element is located in the long open reading frame, within the segment encoding the VP1 capsid protein, the role of the cre in replication is dependent upon its RNA structure and not its protein-coding capacity (12). Mutational analysis and computer folding algorithms suggested that the cre forms a complex stem-loop structure within the positive-strand RNA that is required for initiation of minus-strand RNA synthesis (Fig. (Fig.1)1) (12). FIG. 1. Organization of the subgenomic HRV-14 replicon, ΔP1LucCRE. In ΔP1LucCRE, all of the P1 region was replaced with luciferase coding sequence, except for the 21 nt coding for the carboxy-terminal 7 aa of VP1 (12). The HRV-14 cre was inserted ... Following the identification of the HRV-14 cre, similar internal replication signals were identified within the open reading frames of Theiler's virus, a cardiovirus (9), poliovirus type 1 (PV-1) (5), and, more recently, HRV-2 (4). The latter observations suggest that an internally located cre may be a common feature of the RNA replication schemes of all picornaviruses. The RNA segments comprising these putative cres can be folded into relatively simple stem-loop structures, but the elements differ in terms of their primary nucleotide sequence as well as their location within the open reading frame. In cardioviruses, the cre is located in the VP2 region (9). In contrast, the PV-1 cre is not located in the P1 capsid region, but in the 2C (P2) region (5), while the HRV-2 cre is located within the 2A coding sequence (4). Studies by Paul et al. (17) indicate that the cre acts as the primary template for uridylylation of VPg by the 3Dpol polymerase in vitro. In addition, a recent mutational analysis of the poliovirus polymerase indicates that amino acid residues on the surface of the protein that are essential for uridylylation of VPg are also involved in the interaction of the polymerase with the membrane-bound 3AB precursor protein (10). Taken together, these data suggest that the cre plays a critical role in bringing viral RNA into the replication complex and in initiating VPg uridylylation, the first step in the process of viral RNA replication. Here, we describe experiments aimed at better defining both the sequence and structural requirements for cre function during the replication of HRV-14 RNA. We show that the fully functional cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop structure. By introducing single-base substitutions at each position within the loop sequence, we have determined which nucleotides are essential for replication and which nucleotide substitutions are tolerated without significant degradation of cre function. We further show that the ability of individual mutant cre sequences to support RNA replication is closely correlated with their ability to serve as template for the uridylylation of VPg in an in vitro reaction.
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Implications for secondary structure an
1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:me RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5’ nontranslated region (S’NTR). In other picornaviruses, the S’NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV SNTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the S’NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the SNTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5’NTRs of the cardioviruses and Aphthoviruses.
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Genetic variability within the 5' nontranslated region of hepatitis A virus RNA. Implications for secondary structure and function.
Journal of hepatology, 1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:The RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5' nontranslated region (5'NTR). In other picornaviruses, the 5'NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV 5'NTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the 5'NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the 5'NTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5'NTRs of the cardioviruses and Aphthoviruses.
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Genetic variability within the 5′ nontranslated region of hepatitis A virus RNA: Implications for secondary structure and function
Journal of Hepatology, 1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:Abstract The RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5′ nontranslated region (5′NTR). In other picornaviruses, the 5′NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV 5′NTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the 5′NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the 5′NTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5′NTRs of the cardioviruses and Aphthoviruses.
Ann C. Palmenberg - One of the best experts on this subject based on the ideXlab platform.
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Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation.
Virology, 2001Co-Authors: Cheryl M.t. Dvorak, David J. Hall, Marchel Hill, Michael Riddle, Andrew Pranter, Johnathan Dillman, Michael Deibel, Ann C. PalmenbergAbstract:Abstract Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related Aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173–201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67–76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3Cpro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event.
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Mengovirus and Encephalomyocarditis Virus Poly(C) Tract Lengths Can Affect Virus Growth in Murine Cell Culture
Journal of Virology, 2000Co-Authors: Lee R. Martin, Zane C. Neal, M. Scott Mcbride, Ann C. PalmenbergAbstract:Many virulent Aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5′ untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C44UC10), but supported only substantially diminished and virtually nonlytic growth of vMC24 (C13UC10) and vMC0 short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.
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Tandem mengovirus 5' pseudoknots are linked to viral RNA synthesis, not poly(C)-mediated virulence.
Journal of virology, 1996Co-Authors: Lee R. Martin, Ann C. PalmenbergAbstract:The RNA genomes from the cardioviruses, hepatoviruses, and Aphthoviruses encode two to five tandem pseudoknots within their 5' untranslated regions. These pseudoknots lie adjacent to a pyrimidine-rich sequence, which in cardio- and Aphthoviruses takes the form of a homopolymeric poly(C) tract. Seven deletion mutations within mengovirus pseudoknots PK(B) and PK(C) were created and characterized. tested in tissue culture, mengovirus genomes with alterations in PK(C) were viable but had small plaque phenotypes. Larger plaque revertants were isolated and partially characterized, and each proved to be a second-site pseudorevertant with (unmapped) changes elsewhere in the genome. The infectious PK(C) mutant viruses were highly lethal to mice, and deletions in this motif did not affect mengovirus virulence in the same manner as deletions in the adjacent poly(C) tract. In contrast, deletions in PK(B), or deletions which spanned PK(B) + PK(C), produced nonviable genomes. Cell-free translations directed by any of the altered PK sequences gave normal polyprotein amounts relative to wild-type mengovirus. But viral RNA accumulation during HeLa cell infection was dramatically impaired, even with the least disruptive of the PK(C) changes, suggesting the pseudoknots play an essential though undefined role in RNA synthesis and moreover that an intact PK(B) structure is critical to this function.
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Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors.
Virology, 1992Co-Authors: Ann C. Palmenberg, Griffith D. Parks, David J. Hall, Richard H. Ingraham, Thomas W. Seng, Peter V. PallaiAbstract:Abstract The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5′ nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 213 had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 213, which is conserved in most cardio- and Aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1 D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.
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Attenuation of Mengo virus through genetic engineering of the 5′ noncoding poly(C) tract
Nature, 1990Co-Authors: Gregory M. Duke, Jorge E. Osorio, Ann C. PalmenbergAbstract:THE murine cardioviruses, such as the Mengo and encephalo-myocarditis viruses, and the bovine Aphthoviruses, such as foot-and-mouth disease virus, are distinguished among positive-strand RNA viruses by the presence of long homopolymeric poly(C) tracts within their 5′ noncoding sequences. Although the specific lengths (60–350 bases) and sequence discontinuities (for example, uridine residues) that sometimes disrupt the homopolymer have served to characterize natural viral isolates, the biological function of the poly(C) region has never been clear. We now report that complementary DNA-mediated truncation of the Mengo virus poly(C) tract dramatically attenuates the pathogenicity of the virus in mice. Animals injected with viruses with short tracts not only survived inoculation of up to 50 μg live virus (10^11 plaque-forming units) but consistently produced high titres of neutralizing antibodies, which conferred long-term immunogenic protection from (normally) lethal virus challenge. We propose that analogous synthetic strains of foot and mouth disease virus could serve as the basis for new attenuated vaccines.
Edwin A. Brown - One of the best experts on this subject based on the ideXlab platform.
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Implications for secondary structure an
1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:me RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5’ nontranslated region (S’NTR). In other picornaviruses, the S’NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV SNTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the S’NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the SNTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5’NTRs of the cardioviruses and Aphthoviruses.
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Genetic variability within the 5' nontranslated region of hepatitis A virus RNA. Implications for secondary structure and function.
Journal of hepatology, 1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:The RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5' nontranslated region (5'NTR). In other picornaviruses, the 5'NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV 5'NTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the 5'NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the 5'NTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5'NTRs of the cardioviruses and Aphthoviruses.
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Genetic variability within the 5′ nontranslated region of hepatitis A virus RNA: Implications for secondary structure and function
Journal of Hepatology, 1991Co-Authors: Edwin A. Brown, Stephen P. Day, Robert W. Jansen, Stanley M. LemonAbstract:Abstract The RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5′ nontranslated region (5′NTR). In other picornaviruses, the 5′NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV 5′NTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the 5′NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the 5′NTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5′NTRs of the cardioviruses and Aphthoviruses.
