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James H. Strauss - One of the best experts on this subject based on the ideXlab platform.
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Aura Virus structure suggests that the t 4 organization is a fundamental property of viral structural proteins
Journal of Virology, 2002Co-Authors: Wei Zhang, James H. Strauss, Richard J. Kuhn, Bonnie R Fisher, Norman H Olson, Timothy S. BakerAbstract:Aura and Sindbis Viruses are closely related alphaViruses. Unlike other alphaViruses, Aura Virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form Virus particles. Previous studies on negatively stained Aura Virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura Virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis Virus and Ross River Virus particles. Aura Virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaViruses. The morphology of Aura Virus glycoprotein spikes closely resembles that of Sindbis Virus spikes and is detectably different from that of Ross River Virus spikes. Thus, some aspects of the surface structure of members of the Sindbis Virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River Virus lineage.
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Aura Virus is a new world representative of sindbis like Viruses
Virology, 1995Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, James H. StraussAbstract:Aura Virus is an alphaVirus present in Brazil and Argentina that is serologically related to Sindbis Virus (present throughout the Old World) and to Western equine encephalitis (WEE) Virus (present in the Americas). We have previously shown that WEE is a recombinant Virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like Virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) Virus. We show here that Aura Virus is a Sindbis-like Virus that shares considerable organizational and sequence identity with Sindbis Virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis Virus, and important antigenic epitopes are conserved between the two Viruses. Despite their close relationship, the two Viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis Virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two Viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura Virus. From this we propose that an ancestral Sindbis-like Virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura Virus and a lineage that gave rise to Sindbis Virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Slndbis-like Virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis Viruses distributed throughout the Old World, and in a separate event a Sindbis-like Virus belonging to the same lineage underwent recombination with EEE to give rise to WEE.
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Aura alphaVirus subgenomic RNA is packaged into virions of two sizes.
Journal of virology, 1995Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, Dennis Brown, Matthias König, R Rameriz-mitchel, James H. StraussAbstract:The alphaVirus genome is 11.8 kb in size. During infection, a 4.2-kb subgenomic RNA is also produced. Most alphaViruses package only the genomic RNA into virions, which are enveloped particles with icosahedral symmetry, having a triangulation number (T) = 4. Aura Virus, however, packages both the genomic RNA and the subgenomic RNA into virions. The genomic RNA is primarily packaged into a virion that has a diameter of 72 nm and which appears to be identical to the virions produced by other alphaViruses. The subgenomic RNA is packaged into two major, regular particles with diameters of 72 and 62 nm. The 72-nm-diameter particle appears to be identical in construction to virions containing genomic RNA. The 62-nm-diameter particle probably has T = 3. The large and small Aura virions can be partially separated in sucrose gradients. In addition to these two major classes of particles, there are other particles produced that appear to arise from abortive assembly. From these results and from previous studies of alphaVirus assembly, we suggest that during assembly of alphaVirus nucleocapsids in the infected cell there is a specific initiation event followed by recruitment of additional capsid subunits into the complex, that the triangulation number of the complex is not predetermined but depends upon the size of the RNA and interactions that occur during assembly, and that budding of assembled nucleocapsids results in the acquisition of an envelope containing glycoproteins arranged in a manner determined by the nucleocapsid.
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Subgenomic mRNA of Aura alphaVirus is packaged into virions.
Journal of virology, 1994Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, James H. StraussAbstract:Purified virions of Aura Virus, a South American alphaVirus related to Sindbis Virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in Virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the Virus. Although 26S RNA is present in the cytosol of all cells infected by alphaViruses, this is the first report of incorporation of the subgenomic RNA into alphaVirus particles. Packaging of the Aura Virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis Virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura Virus than in cells infected by Sindbis Virus. Packaging of the Aura Virus subgenomic RNA, but not those of other alphaViruses, suggests that Aura Virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the Virus remains to be determined.
Shailly Tomar - One of the best experts on this subject based on the ideXlab platform.
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evaluation of antiviral activity of piperazine against chikungunya Virus targeting hydrophobic pocket of alphaVirus capsid protein
Antiviral Research, 2017Co-Authors: Megha Aggarwal, Paban Kumar Dash, Manmohan Parida, Ramanjit Kaur, Amrita Saha, Rajat Mudgal, Ravi Yadav, Pravindra Kumar, Shailly TomarAbstract:Small heterocyclic molecules such as piperazine are potential pharmacotherapeutic agents and binding of these molecules to the hydrophobic pocket of capsid protein (CP) offers a new perspective for therapeutic intervention. Here, we report the crystal structure of CP from Aura Virus (AVCP) in complex with piperazine at 2.2 A resolution. Piperazine binds to the conserved hydrophobic pocket of CP where dioxane based antivirals bind. Comparative structural studies of the piperazine-bound AVCP structure with the apo, active and dioxane-bound AVCP structures provide insights into the conformational variations in the pocket. Additionally, the molecular docking studies showed that piperazine binds into the hydrophobic pocket of Chikungunya Virus CP (CVCP) with more affinity than with AVCP. Furthermore, the antiviral activity of piperazine against Chikungunya Virus (CHIKV) was investigated by plaque reduction and immunofluorescence assays. The AVCP-piperazine complex may serve as a lead scaffold for structure-based design of piperazine derivatives as alphaviral inhibitors. The antiviral properties of piperazine provide its usefulness for further investigations towards the development of piperazine based anti-alphaviral drugs.
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Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid- Glycoprotein Molecular Contacts
2016Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphaVirus life cycle that is essential to Virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura Virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura Virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis Virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura Virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimic
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CP-glycoproteins interaction along with analysis of different amino acids in cdE2.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:(A) Representation of the crystal structure of AVCP-dioxane complex and the homology models of E1 and E2 glycoproteins fitted into the cryo-EM electron density map of VEEV (EMDB ID: 5275). AVCP, E1 and E2 are shown in green, yellow and pink respectively; bound dioxane is shown in red while the cryo-EM density map is denoted by gray; (B) Structural alignment of cdE2 predicted structure from Aura Virus (pink) and VEEV (blue) showing Tyr401 residue oriented towards other helix away from CP and the disulfide bond (yellow) within the helix-loop-helix motif of E2. The Pro405 interaction with the hydrophobic pocket present on the surface of the AVCP crystal structure is also shown; (C) Cartoon view of the AVCP-glycoproteins interaction. Binding of Aura Virus E2 and E1 cytoplasmic tails to different pockets (P1 and P2 respectively) found in close proximity at the surface of AVCP. E2 shows the helix-loop-helix structural architecture and E1 has a helical structure that interacts with AVCP. The loop separating both interacting pockets is shown in red color; (D) The critical polar interactions between E2 and capsid as well as within the helix-loop-helix motif of cdE2 are shown in dotted lines. E2 residues are shown in pink while the AVCP residues are displayed in green. The interacting residues are shown as sticks.
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Multiple sequence alignment of the cytoplasmic domain of E2 (cdE2) from different alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The secondary structure of 33 residues from Aura Virus cdE2 displays a helix-loop-helix structure. Highly conserved residues are in red background. The circles under residues denote the disulfide bridge forming Cys residues, squares indicate the residues responsible for polar interactions within the E2 helix-loop-helix motif and the triangles represent the residues of E2 making polar interactions with CP. cdE2 sequences used for alignment are: Sindbis (SinVcdE2), Western equine encephalitis (WEEVcdE2), Aura (AuraVcdE2), Chikungunya (ChikVcdE2), Venezuelan equine encephalitis (VEEVcdE2), Ross River (RRVcdE2) and Semliki Forest (SFVcdE2).
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Multiple sequence alignment of AVCP with CPs from other alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The conserved residues are shown in red background. The circles under the amino acids indicate the hydrophobic pocket residues interacting with the dioxane molecule while the triangles denote the catalytic triad residues. The residues forming salt bridges are denoted by squares below the residues. The motif responsible for specificity in encapsidation is highlighted in rectangular box 1. Interdomain flexible loop residues are shown in box 2 whereas the flexible loop separating the two pockets for E1 and E2 binding is shown in box 3. Capsid protein sequences used for alignment are: Aura Virus (AVCP), Sindbis (SCP), Ross River (RRCP), Semliki Forest (SFCP), Chikungunya (ChikVCP), Venezuelan equine encephalitis (VEEVCP) and Western equine encephalitis (WEEVCP).
Megha Aggarwal - One of the best experts on this subject based on the ideXlab platform.
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evaluation of antiviral activity of piperazine against chikungunya Virus targeting hydrophobic pocket of alphaVirus capsid protein
Antiviral Research, 2017Co-Authors: Megha Aggarwal, Paban Kumar Dash, Manmohan Parida, Ramanjit Kaur, Amrita Saha, Rajat Mudgal, Ravi Yadav, Pravindra Kumar, Shailly TomarAbstract:Small heterocyclic molecules such as piperazine are potential pharmacotherapeutic agents and binding of these molecules to the hydrophobic pocket of capsid protein (CP) offers a new perspective for therapeutic intervention. Here, we report the crystal structure of CP from Aura Virus (AVCP) in complex with piperazine at 2.2 A resolution. Piperazine binds to the conserved hydrophobic pocket of CP where dioxane based antivirals bind. Comparative structural studies of the piperazine-bound AVCP structure with the apo, active and dioxane-bound AVCP structures provide insights into the conformational variations in the pocket. Additionally, the molecular docking studies showed that piperazine binds into the hydrophobic pocket of Chikungunya Virus CP (CVCP) with more affinity than with AVCP. Furthermore, the antiviral activity of piperazine against Chikungunya Virus (CHIKV) was investigated by plaque reduction and immunofluorescence assays. The AVCP-piperazine complex may serve as a lead scaffold for structure-based design of piperazine derivatives as alphaviral inhibitors. The antiviral properties of piperazine provide its usefulness for further investigations towards the development of piperazine based anti-alphaviral drugs.
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Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid- Glycoprotein Molecular Contacts
2016Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphaVirus life cycle that is essential to Virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura Virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura Virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis Virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura Virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimic
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CP-glycoproteins interaction along with analysis of different amino acids in cdE2.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:(A) Representation of the crystal structure of AVCP-dioxane complex and the homology models of E1 and E2 glycoproteins fitted into the cryo-EM electron density map of VEEV (EMDB ID: 5275). AVCP, E1 and E2 are shown in green, yellow and pink respectively; bound dioxane is shown in red while the cryo-EM density map is denoted by gray; (B) Structural alignment of cdE2 predicted structure from Aura Virus (pink) and VEEV (blue) showing Tyr401 residue oriented towards other helix away from CP and the disulfide bond (yellow) within the helix-loop-helix motif of E2. The Pro405 interaction with the hydrophobic pocket present on the surface of the AVCP crystal structure is also shown; (C) Cartoon view of the AVCP-glycoproteins interaction. Binding of Aura Virus E2 and E1 cytoplasmic tails to different pockets (P1 and P2 respectively) found in close proximity at the surface of AVCP. E2 shows the helix-loop-helix structural architecture and E1 has a helical structure that interacts with AVCP. The loop separating both interacting pockets is shown in red color; (D) The critical polar interactions between E2 and capsid as well as within the helix-loop-helix motif of cdE2 are shown in dotted lines. E2 residues are shown in pink while the AVCP residues are displayed in green. The interacting residues are shown as sticks.
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Multiple sequence alignment of the cytoplasmic domain of E2 (cdE2) from different alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The secondary structure of 33 residues from Aura Virus cdE2 displays a helix-loop-helix structure. Highly conserved residues are in red background. The circles under residues denote the disulfide bridge forming Cys residues, squares indicate the residues responsible for polar interactions within the E2 helix-loop-helix motif and the triangles represent the residues of E2 making polar interactions with CP. cdE2 sequences used for alignment are: Sindbis (SinVcdE2), Western equine encephalitis (WEEVcdE2), Aura (AuraVcdE2), Chikungunya (ChikVcdE2), Venezuelan equine encephalitis (VEEVcdE2), Ross River (RRVcdE2) and Semliki Forest (SFVcdE2).
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Multiple sequence alignment of AVCP with CPs from other alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The conserved residues are shown in red background. The circles under the amino acids indicate the hydrophobic pocket residues interacting with the dioxane molecule while the triangles denote the catalytic triad residues. The residues forming salt bridges are denoted by squares below the residues. The motif responsible for specificity in encapsidation is highlighted in rectangular box 1. Interdomain flexible loop residues are shown in box 2 whereas the flexible loop separating the two pockets for E1 and E2 binding is shown in box 3. Capsid protein sequences used for alignment are: Aura Virus (AVCP), Sindbis (SCP), Ross River (RRCP), Semliki Forest (SFCP), Chikungunya (ChikVCP), Venezuelan equine encephalitis (VEEVCP) and Western equine encephalitis (WEEVCP).
Tillmann Rümenapf - One of the best experts on this subject based on the ideXlab platform.
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Aura Virus is a new world representative of sindbis like Viruses
Virology, 1995Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, James H. StraussAbstract:Aura Virus is an alphaVirus present in Brazil and Argentina that is serologically related to Sindbis Virus (present throughout the Old World) and to Western equine encephalitis (WEE) Virus (present in the Americas). We have previously shown that WEE is a recombinant Virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like Virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) Virus. We show here that Aura Virus is a Sindbis-like Virus that shares considerable organizational and sequence identity with Sindbis Virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis Virus, and important antigenic epitopes are conserved between the two Viruses. Despite their close relationship, the two Viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis Virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two Viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura Virus. From this we propose that an ancestral Sindbis-like Virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura Virus and a lineage that gave rise to Sindbis Virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Slndbis-like Virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis Viruses distributed throughout the Old World, and in a separate event a Sindbis-like Virus belonging to the same lineage underwent recombination with EEE to give rise to WEE.
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Aura alphaVirus subgenomic RNA is packaged into virions of two sizes.
Journal of virology, 1995Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, Dennis Brown, Matthias König, R Rameriz-mitchel, James H. StraussAbstract:The alphaVirus genome is 11.8 kb in size. During infection, a 4.2-kb subgenomic RNA is also produced. Most alphaViruses package only the genomic RNA into virions, which are enveloped particles with icosahedral symmetry, having a triangulation number (T) = 4. Aura Virus, however, packages both the genomic RNA and the subgenomic RNA into virions. The genomic RNA is primarily packaged into a virion that has a diameter of 72 nm and which appears to be identical to the virions produced by other alphaViruses. The subgenomic RNA is packaged into two major, regular particles with diameters of 72 and 62 nm. The 72-nm-diameter particle appears to be identical in construction to virions containing genomic RNA. The 62-nm-diameter particle probably has T = 3. The large and small Aura virions can be partially separated in sucrose gradients. In addition to these two major classes of particles, there are other particles produced that appear to arise from abortive assembly. From these results and from previous studies of alphaVirus assembly, we suggest that during assembly of alphaVirus nucleocapsids in the infected cell there is a specific initiation event followed by recruitment of additional capsid subunits into the complex, that the triangulation number of the complex is not predetermined but depends upon the size of the RNA and interactions that occur during assembly, and that budding of assembled nucleocapsids results in the acquisition of an envelope containing glycoproteins arranged in a manner determined by the nucleocapsid.
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Subgenomic mRNA of Aura alphaVirus is packaged into virions.
Journal of virology, 1994Co-Authors: Tillmann Rümenapf, Ellen G. Strauss, James H. StraussAbstract:Purified virions of Aura Virus, a South American alphaVirus related to Sindbis Virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in Virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the Virus. Although 26S RNA is present in the cytosol of all cells infected by alphaViruses, this is the first report of incorporation of the subgenomic RNA into alphaVirus particles. Packaging of the Aura Virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis Virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura Virus than in cells infected by Sindbis Virus. Packaging of the Aura Virus subgenomic RNA, but not those of other alphaViruses, suggests that Aura Virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the Virus remains to be determined.
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Copyright © 1994, American Society for Microbiology Subgenomic mRNA of Aura AlphaVirus Is Packaged
1993Co-Authors: Into Virions, Tillmann Rümenapf, Ellen G. Strauss, H. StraussAbstract:Purified virions of Aura Virus, a South American alphaVirus related to Sindbis Virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in Virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the Virus. Although 26S RNA is present in the cytosol of all cells infected by alphaViruses, this is the first report of incorporation of the subgenomic RNA into alphaVirus particles. Packaging of the Aura Virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis Virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura Virus than in cells infected by Sindbis Virus. Packaging of the Aura Virus subgenomic RNA, but not those of other alphaViruses, suggest
Richard J. Kuhn - One of the best experts on this subject based on the ideXlab platform.
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Crystal Structure of Aura Virus Capsid Protease and Its Complex with Dioxane: New Insights into Capsid- Glycoprotein Molecular Contacts
2016Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphaVirus life cycle that is essential to Virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura Virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura Virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis Virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura Virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimic
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CP-glycoproteins interaction along with analysis of different amino acids in cdE2.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:(A) Representation of the crystal structure of AVCP-dioxane complex and the homology models of E1 and E2 glycoproteins fitted into the cryo-EM electron density map of VEEV (EMDB ID: 5275). AVCP, E1 and E2 are shown in green, yellow and pink respectively; bound dioxane is shown in red while the cryo-EM density map is denoted by gray; (B) Structural alignment of cdE2 predicted structure from Aura Virus (pink) and VEEV (blue) showing Tyr401 residue oriented towards other helix away from CP and the disulfide bond (yellow) within the helix-loop-helix motif of E2. The Pro405 interaction with the hydrophobic pocket present on the surface of the AVCP crystal structure is also shown; (C) Cartoon view of the AVCP-glycoproteins interaction. Binding of Aura Virus E2 and E1 cytoplasmic tails to different pockets (P1 and P2 respectively) found in close proximity at the surface of AVCP. E2 shows the helix-loop-helix structural architecture and E1 has a helical structure that interacts with AVCP. The loop separating both interacting pockets is shown in red color; (D) The critical polar interactions between E2 and capsid as well as within the helix-loop-helix motif of cdE2 are shown in dotted lines. E2 residues are shown in pink while the AVCP residues are displayed in green. The interacting residues are shown as sticks.
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Multiple sequence alignment of the cytoplasmic domain of E2 (cdE2) from different alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The secondary structure of 33 residues from Aura Virus cdE2 displays a helix-loop-helix structure. Highly conserved residues are in red background. The circles under residues denote the disulfide bridge forming Cys residues, squares indicate the residues responsible for polar interactions within the E2 helix-loop-helix motif and the triangles represent the residues of E2 making polar interactions with CP. cdE2 sequences used for alignment are: Sindbis (SinVcdE2), Western equine encephalitis (WEEVcdE2), Aura (AuraVcdE2), Chikungunya (ChikVcdE2), Venezuelan equine encephalitis (VEEVcdE2), Ross River (RRVcdE2) and Semliki Forest (SFVcdE2).
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Multiple sequence alignment of AVCP with CPs from other alphaViruses.
2013Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The conserved residues are shown in red background. The circles under the amino acids indicate the hydrophobic pocket residues interacting with the dioxane molecule while the triangles denote the catalytic triad residues. The residues forming salt bridges are denoted by squares below the residues. The motif responsible for specificity in encapsidation is highlighted in rectangular box 1. Interdomain flexible loop residues are shown in box 2 whereas the flexible loop separating the two pockets for E1 and E2 binding is shown in box 3. Capsid protein sequences used for alignment are: Aura Virus (AVCP), Sindbis (SCP), Ross River (RRCP), Semliki Forest (SFCP), Chikungunya (ChikVCP), Venezuelan equine encephalitis (VEEVCP) and Western equine encephalitis (WEEVCP).
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crystal structure of Aura Virus capsid protease and its complex with dioxane new insights into capsid glycoprotein molecular contacts
PLOS ONE, 2012Co-Authors: Megha Aggarwal, Pravindra Kumar, Richard J. Kuhn, Satya Tapas, Anjul Siwach, Shailly TomarAbstract:The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphaVirus life cycle that is essential to Virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura Virus and its complex with dioxane were determined at 1.81 and 1.98 A resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura Virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis Virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura Virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2.
