The Experts below are selected from a list of 49509 Experts worldwide ranked by ideXlab platform
Margaret R. Macdonald - One of the best experts on this subject based on the ideXlab platform.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of Virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Alphaviruses are important human and animal pathogens, causing fever, rash, arthritis, encephalitis, and death (reviewed in reference 18). These enveloped positive-strand RNA viruses cycle in nature through small vertebrate reservoir hosts and invertebrate vectors, most commonly mosquitoes. Infection of larger mammals, including humans, through the bite of an infected mosquito can lead to severe disease, for which there is no specific therapy. Viruses in the Alphavirus genus share common genomic organization and replication strategies, and much has been learned through study of Sindbis virus (SINV), the prototype virus in this genus. SINV, like the other alphaviruses, enters cells by receptor-mediated endocytosis and after fusion in the endosome releases the positive-strand RNA genome, which is translated in the cytoplasm to generate a nonstructural polyProtein, P123 (reviewed in reference 36). Translational readthrough of an opal termination codon also results in the production of the polyProtein P1234. The activity of a cysteine protease residing in nsP2 mediates polyProtein processing, which results in the generation of P123 and nonstructural Protein 4 (nsP4), the viral RNA-dependent RNA polymerase. Together, P123 and nsP4 produce a full-length negative-strand copy of the genomic RNA. Further regulated sequential processing of the polyProtein results in replication complexes active in both positive and negative strand RNA synthesis. After complete proteolytic processing, replication complexes containing the individual nsP1, nsP2, nsP3, and nsP4 Proteins are capable of only positive-strand RNA production, including both genomic RNA and a subgenomic RNA colinear with the 3′ end of the genome and encoding the viral structural Proteins. The roles of the individual viral Proteins in viral RNA replication have been investigated by examination of temperature-sensitive mutants, as well as targeted mutagenesis (reviewed in reference 20). nsP1 interacts with cellular membranes and plays a role in capping the newly generated genomic and subgenomic transcripts. In addition to its proteolytic activity, nsP2 possesses nucleotriphosphatase and helicase activities and is involved in regulation of RNA synthesis. The nsP2 Protein of SINV, as well as that of Semliki Forest virus, also has known activity in the alteration of host cellular processes, such as the shutoff of host translation and transcription (10, 12, 14, 16, 29). Mutational analysis has demonstrated a role for nsP3 in RNA synthesis, although the precise function is not understood. Several recent nsP3 interactome studies (6, 11, 17) have identified nsP3-associated cellular factors that suggest possible roles for this viral Protein in modulation of host cellular pathways. The alphavirus nsP4 is the RNA-dependent RNA polymerase with recent biochemical studies confirming and characterizing its activity (32, 38, 39). Although roles in RNA replication for the virus-encoded nonstructural Proteins (nsPs) have been elucidated (reviewed in references 20 and 36), little is known regarding how host factors may participate in the replication process. To address this, we and others have taken proteomic approaches to identify candidate host factors that may be involved in SINV replication. Using SINV expressing nsPs tagged in frame with the green fluorescent Protein (GFP), host Proteins that interact with nsP2 (1) and nsP3 (6, 11, 17) have been identified. The results from those studies allow for the generation of hypotheses as to how SINV utilizes host factors for viral replication processes. Many alphaviruses, including SINV, express P123 at levels higher than nsP4, the RNA-dependent RNA polymerase, due to an opal termination codon between nsP3 and nsP4 (35) and due to rapid degradation of nsP4 (8). We reasoned, therefore, that nsP1, nsP2, and nsP3 likely have multiple functions within the infected cell and that host Proteins previously identified as interacting with nsP2 and nsP3 may play roles in important processes that might not be directly related to replication of the viral RNA. In fact, evidence has recently been found for multiple types of nsP3-containing complexes in SINV-infected cells (17), suggesting that the functions of these viral Proteins and their interacting host Proteins may be multifaceted. We therefore undertook studies to identify host factors that interact with nsP4, the viral RNA-dependent RNA polymerase, to identify candidate host factors that might participate more directly in viral RNA replication. In the present study we isolated a Flag-tagged version of the viral RNA-dependent RNA polymerase nsP4 from infected vertebrate cells at two different times of infection, a late time (12 h) and an earlier time (6 h), and identified the interacting host Proteins by mass spectrometry. Some Proteins, e.g., Protein arginine methyltransferase 5 (PRMT5) and unc-84 homology B (UNC84), were detected in association with nsP4 only at the earlier time point. Others, e.g., adenine nucleotide translocator 1 (ANT1) and F1-ATPase, were observed only at the late time point. Some host factors, e.g., methylosome Protein 50 (MEP50), heat shock Protein 90 (HSP90), and the Ras-GTPase-activating Protein SH3-domain-binding Protein 1 (G3BP1) and G3BP2, were associated with nsP4 at both the early and late time points. The identified Proteins may play direct roles in viral RNA replication, or may be participating in host responses aimed at limiting viral replication. Two Proteins, G3BP1 and G3BP2, identified in nsP4-containing complexes were also identified in nsP2 (1)- and nsP3 (6, 11, 17)-containing complexes in prior studies. We demonstrate here a role for these Proteins in limiting SINV replication events.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host Proteins. Of these, 10 Proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host Proteins may be required for the multiple functions carried out by nsP4. Two related Proteins found in association with nsP4 at both times of infection, GTPase-activating Protein (SH3 domain) binding Protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Ileana M Cristea - One of the best experts on this subject based on the ideXlab platform.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of Virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Alphaviruses are important human and animal pathogens, causing fever, rash, arthritis, encephalitis, and death (reviewed in reference 18). These enveloped positive-strand RNA viruses cycle in nature through small vertebrate reservoir hosts and invertebrate vectors, most commonly mosquitoes. Infection of larger mammals, including humans, through the bite of an infected mosquito can lead to severe disease, for which there is no specific therapy. Viruses in the Alphavirus genus share common genomic organization and replication strategies, and much has been learned through study of Sindbis virus (SINV), the prototype virus in this genus. SINV, like the other alphaviruses, enters cells by receptor-mediated endocytosis and after fusion in the endosome releases the positive-strand RNA genome, which is translated in the cytoplasm to generate a nonstructural polyProtein, P123 (reviewed in reference 36). Translational readthrough of an opal termination codon also results in the production of the polyProtein P1234. The activity of a cysteine protease residing in nsP2 mediates polyProtein processing, which results in the generation of P123 and nonstructural Protein 4 (nsP4), the viral RNA-dependent RNA polymerase. Together, P123 and nsP4 produce a full-length negative-strand copy of the genomic RNA. Further regulated sequential processing of the polyProtein results in replication complexes active in both positive and negative strand RNA synthesis. After complete proteolytic processing, replication complexes containing the individual nsP1, nsP2, nsP3, and nsP4 Proteins are capable of only positive-strand RNA production, including both genomic RNA and a subgenomic RNA colinear with the 3′ end of the genome and encoding the viral structural Proteins. The roles of the individual viral Proteins in viral RNA replication have been investigated by examination of temperature-sensitive mutants, as well as targeted mutagenesis (reviewed in reference 20). nsP1 interacts with cellular membranes and plays a role in capping the newly generated genomic and subgenomic transcripts. In addition to its proteolytic activity, nsP2 possesses nucleotriphosphatase and helicase activities and is involved in regulation of RNA synthesis. The nsP2 Protein of SINV, as well as that of Semliki Forest virus, also has known activity in the alteration of host cellular processes, such as the shutoff of host translation and transcription (10, 12, 14, 16, 29). Mutational analysis has demonstrated a role for nsP3 in RNA synthesis, although the precise function is not understood. Several recent nsP3 interactome studies (6, 11, 17) have identified nsP3-associated cellular factors that suggest possible roles for this viral Protein in modulation of host cellular pathways. The alphavirus nsP4 is the RNA-dependent RNA polymerase with recent biochemical studies confirming and characterizing its activity (32, 38, 39). Although roles in RNA replication for the virus-encoded nonstructural Proteins (nsPs) have been elucidated (reviewed in references 20 and 36), little is known regarding how host factors may participate in the replication process. To address this, we and others have taken proteomic approaches to identify candidate host factors that may be involved in SINV replication. Using SINV expressing nsPs tagged in frame with the green fluorescent Protein (GFP), host Proteins that interact with nsP2 (1) and nsP3 (6, 11, 17) have been identified. The results from those studies allow for the generation of hypotheses as to how SINV utilizes host factors for viral replication processes. Many alphaviruses, including SINV, express P123 at levels higher than nsP4, the RNA-dependent RNA polymerase, due to an opal termination codon between nsP3 and nsP4 (35) and due to rapid degradation of nsP4 (8). We reasoned, therefore, that nsP1, nsP2, and nsP3 likely have multiple functions within the infected cell and that host Proteins previously identified as interacting with nsP2 and nsP3 may play roles in important processes that might not be directly related to replication of the viral RNA. In fact, evidence has recently been found for multiple types of nsP3-containing complexes in SINV-infected cells (17), suggesting that the functions of these viral Proteins and their interacting host Proteins may be multifaceted. We therefore undertook studies to identify host factors that interact with nsP4, the viral RNA-dependent RNA polymerase, to identify candidate host factors that might participate more directly in viral RNA replication. In the present study we isolated a Flag-tagged version of the viral RNA-dependent RNA polymerase nsP4 from infected vertebrate cells at two different times of infection, a late time (12 h) and an earlier time (6 h), and identified the interacting host Proteins by mass spectrometry. Some Proteins, e.g., Protein arginine methyltransferase 5 (PRMT5) and unc-84 homology B (UNC84), were detected in association with nsP4 only at the earlier time point. Others, e.g., adenine nucleotide translocator 1 (ANT1) and F1-ATPase, were observed only at the late time point. Some host factors, e.g., methylosome Protein 50 (MEP50), heat shock Protein 90 (HSP90), and the Ras-GTPase-activating Protein SH3-domain-binding Protein 1 (G3BP1) and G3BP2, were associated with nsP4 at both the early and late time points. The identified Proteins may play direct roles in viral RNA replication, or may be participating in host responses aimed at limiting viral replication. Two Proteins, G3BP1 and G3BP2, identified in nsP4-containing complexes were also identified in nsP2 (1)- and nsP3 (6, 11, 17)-containing complexes in prior studies. We demonstrate here a role for these Proteins in limiting SINV replication events.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host Proteins. Of these, 10 Proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host Proteins may be required for the multiple functions carried out by nsP4. Two related Proteins found in association with nsP4 at both times of infection, GTPase-activating Protein (SH3 domain) binding Protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Charles M Rice - One of the best experts on this subject based on the ideXlab platform.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of Virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Alphaviruses are important human and animal pathogens, causing fever, rash, arthritis, encephalitis, and death (reviewed in reference 18). These enveloped positive-strand RNA viruses cycle in nature through small vertebrate reservoir hosts and invertebrate vectors, most commonly mosquitoes. Infection of larger mammals, including humans, through the bite of an infected mosquito can lead to severe disease, for which there is no specific therapy. Viruses in the Alphavirus genus share common genomic organization and replication strategies, and much has been learned through study of Sindbis virus (SINV), the prototype virus in this genus. SINV, like the other alphaviruses, enters cells by receptor-mediated endocytosis and after fusion in the endosome releases the positive-strand RNA genome, which is translated in the cytoplasm to generate a nonstructural polyProtein, P123 (reviewed in reference 36). Translational readthrough of an opal termination codon also results in the production of the polyProtein P1234. The activity of a cysteine protease residing in nsP2 mediates polyProtein processing, which results in the generation of P123 and nonstructural Protein 4 (nsP4), the viral RNA-dependent RNA polymerase. Together, P123 and nsP4 produce a full-length negative-strand copy of the genomic RNA. Further regulated sequential processing of the polyProtein results in replication complexes active in both positive and negative strand RNA synthesis. After complete proteolytic processing, replication complexes containing the individual nsP1, nsP2, nsP3, and nsP4 Proteins are capable of only positive-strand RNA production, including both genomic RNA and a subgenomic RNA colinear with the 3′ end of the genome and encoding the viral structural Proteins. The roles of the individual viral Proteins in viral RNA replication have been investigated by examination of temperature-sensitive mutants, as well as targeted mutagenesis (reviewed in reference 20). nsP1 interacts with cellular membranes and plays a role in capping the newly generated genomic and subgenomic transcripts. In addition to its proteolytic activity, nsP2 possesses nucleotriphosphatase and helicase activities and is involved in regulation of RNA synthesis. The nsP2 Protein of SINV, as well as that of Semliki Forest virus, also has known activity in the alteration of host cellular processes, such as the shutoff of host translation and transcription (10, 12, 14, 16, 29). Mutational analysis has demonstrated a role for nsP3 in RNA synthesis, although the precise function is not understood. Several recent nsP3 interactome studies (6, 11, 17) have identified nsP3-associated cellular factors that suggest possible roles for this viral Protein in modulation of host cellular pathways. The alphavirus nsP4 is the RNA-dependent RNA polymerase with recent biochemical studies confirming and characterizing its activity (32, 38, 39). Although roles in RNA replication for the virus-encoded nonstructural Proteins (nsPs) have been elucidated (reviewed in references 20 and 36), little is known regarding how host factors may participate in the replication process. To address this, we and others have taken proteomic approaches to identify candidate host factors that may be involved in SINV replication. Using SINV expressing nsPs tagged in frame with the green fluorescent Protein (GFP), host Proteins that interact with nsP2 (1) and nsP3 (6, 11, 17) have been identified. The results from those studies allow for the generation of hypotheses as to how SINV utilizes host factors for viral replication processes. Many alphaviruses, including SINV, express P123 at levels higher than nsP4, the RNA-dependent RNA polymerase, due to an opal termination codon between nsP3 and nsP4 (35) and due to rapid degradation of nsP4 (8). We reasoned, therefore, that nsP1, nsP2, and nsP3 likely have multiple functions within the infected cell and that host Proteins previously identified as interacting with nsP2 and nsP3 may play roles in important processes that might not be directly related to replication of the viral RNA. In fact, evidence has recently been found for multiple types of nsP3-containing complexes in SINV-infected cells (17), suggesting that the functions of these viral Proteins and their interacting host Proteins may be multifaceted. We therefore undertook studies to identify host factors that interact with nsP4, the viral RNA-dependent RNA polymerase, to identify candidate host factors that might participate more directly in viral RNA replication. In the present study we isolated a Flag-tagged version of the viral RNA-dependent RNA polymerase nsP4 from infected vertebrate cells at two different times of infection, a late time (12 h) and an earlier time (6 h), and identified the interacting host Proteins by mass spectrometry. Some Proteins, e.g., Protein arginine methyltransferase 5 (PRMT5) and unc-84 homology B (UNC84), were detected in association with nsP4 only at the earlier time point. Others, e.g., adenine nucleotide translocator 1 (ANT1) and F1-ATPase, were observed only at the late time point. Some host factors, e.g., methylosome Protein 50 (MEP50), heat shock Protein 90 (HSP90), and the Ras-GTPase-activating Protein SH3-domain-binding Protein 1 (G3BP1) and G3BP2, were associated with nsP4 at both the early and late time points. The identified Proteins may play direct roles in viral RNA replication, or may be participating in host responses aimed at limiting viral replication. Two Proteins, G3BP1 and G3BP2, identified in nsP4-containing complexes were also identified in nsP2 (1)- and nsP3 (6, 11, 17)-containing complexes in prior studies. We demonstrate here a role for these Proteins in limiting SINV replication events.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host Proteins. Of these, 10 Proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host Proteins may be required for the multiple functions carried out by nsP4. Two related Proteins found in association with nsP4 at both times of infection, GTPase-activating Protein (SH3 domain) binding Protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Kelly R Molloy - One of the best experts on this subject based on the ideXlab platform.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of Virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Alphaviruses are important human and animal pathogens, causing fever, rash, arthritis, encephalitis, and death (reviewed in reference 18). These enveloped positive-strand RNA viruses cycle in nature through small vertebrate reservoir hosts and invertebrate vectors, most commonly mosquitoes. Infection of larger mammals, including humans, through the bite of an infected mosquito can lead to severe disease, for which there is no specific therapy. Viruses in the Alphavirus genus share common genomic organization and replication strategies, and much has been learned through study of Sindbis virus (SINV), the prototype virus in this genus. SINV, like the other alphaviruses, enters cells by receptor-mediated endocytosis and after fusion in the endosome releases the positive-strand RNA genome, which is translated in the cytoplasm to generate a nonstructural polyProtein, P123 (reviewed in reference 36). Translational readthrough of an opal termination codon also results in the production of the polyProtein P1234. The activity of a cysteine protease residing in nsP2 mediates polyProtein processing, which results in the generation of P123 and nonstructural Protein 4 (nsP4), the viral RNA-dependent RNA polymerase. Together, P123 and nsP4 produce a full-length negative-strand copy of the genomic RNA. Further regulated sequential processing of the polyProtein results in replication complexes active in both positive and negative strand RNA synthesis. After complete proteolytic processing, replication complexes containing the individual nsP1, nsP2, nsP3, and nsP4 Proteins are capable of only positive-strand RNA production, including both genomic RNA and a subgenomic RNA colinear with the 3′ end of the genome and encoding the viral structural Proteins. The roles of the individual viral Proteins in viral RNA replication have been investigated by examination of temperature-sensitive mutants, as well as targeted mutagenesis (reviewed in reference 20). nsP1 interacts with cellular membranes and plays a role in capping the newly generated genomic and subgenomic transcripts. In addition to its proteolytic activity, nsP2 possesses nucleotriphosphatase and helicase activities and is involved in regulation of RNA synthesis. The nsP2 Protein of SINV, as well as that of Semliki Forest virus, also has known activity in the alteration of host cellular processes, such as the shutoff of host translation and transcription (10, 12, 14, 16, 29). Mutational analysis has demonstrated a role for nsP3 in RNA synthesis, although the precise function is not understood. Several recent nsP3 interactome studies (6, 11, 17) have identified nsP3-associated cellular factors that suggest possible roles for this viral Protein in modulation of host cellular pathways. The alphavirus nsP4 is the RNA-dependent RNA polymerase with recent biochemical studies confirming and characterizing its activity (32, 38, 39). Although roles in RNA replication for the virus-encoded nonstructural Proteins (nsPs) have been elucidated (reviewed in references 20 and 36), little is known regarding how host factors may participate in the replication process. To address this, we and others have taken proteomic approaches to identify candidate host factors that may be involved in SINV replication. Using SINV expressing nsPs tagged in frame with the green fluorescent Protein (GFP), host Proteins that interact with nsP2 (1) and nsP3 (6, 11, 17) have been identified. The results from those studies allow for the generation of hypotheses as to how SINV utilizes host factors for viral replication processes. Many alphaviruses, including SINV, express P123 at levels higher than nsP4, the RNA-dependent RNA polymerase, due to an opal termination codon between nsP3 and nsP4 (35) and due to rapid degradation of nsP4 (8). We reasoned, therefore, that nsP1, nsP2, and nsP3 likely have multiple functions within the infected cell and that host Proteins previously identified as interacting with nsP2 and nsP3 may play roles in important processes that might not be directly related to replication of the viral RNA. In fact, evidence has recently been found for multiple types of nsP3-containing complexes in SINV-infected cells (17), suggesting that the functions of these viral Proteins and their interacting host Proteins may be multifaceted. We therefore undertook studies to identify host factors that interact with nsP4, the viral RNA-dependent RNA polymerase, to identify candidate host factors that might participate more directly in viral RNA replication. In the present study we isolated a Flag-tagged version of the viral RNA-dependent RNA polymerase nsP4 from infected vertebrate cells at two different times of infection, a late time (12 h) and an earlier time (6 h), and identified the interacting host Proteins by mass spectrometry. Some Proteins, e.g., Protein arginine methyltransferase 5 (PRMT5) and unc-84 homology B (UNC84), were detected in association with nsP4 only at the earlier time point. Others, e.g., adenine nucleotide translocator 1 (ANT1) and F1-ATPase, were observed only at the late time point. Some host factors, e.g., methylosome Protein 50 (MEP50), heat shock Protein 90 (HSP90), and the Ras-GTPase-activating Protein SH3-domain-binding Protein 1 (G3BP1) and G3BP2, were associated with nsP4 at both the early and late time points. The identified Proteins may play direct roles in viral RNA replication, or may be participating in host responses aimed at limiting viral replication. Two Proteins, G3BP1 and G3BP2, identified in nsP4-containing complexes were also identified in nsP2 (1)- and nsP3 (6, 11, 17)-containing complexes in prior studies. We demonstrate here a role for these Proteins in limiting SINV replication events.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host Proteins. Of these, 10 Proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host Proteins may be required for the multiple functions carried out by nsP4. Two related Proteins found in association with nsP4 at both times of infection, GTPase-activating Protein (SH3 domain) binding Protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Heather Rozjabek - One of the best experts on this subject based on the ideXlab platform.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of Virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Alphaviruses are important human and animal pathogens, causing fever, rash, arthritis, encephalitis, and death (reviewed in reference 18). These enveloped positive-strand RNA viruses cycle in nature through small vertebrate reservoir hosts and invertebrate vectors, most commonly mosquitoes. Infection of larger mammals, including humans, through the bite of an infected mosquito can lead to severe disease, for which there is no specific therapy. Viruses in the Alphavirus genus share common genomic organization and replication strategies, and much has been learned through study of Sindbis virus (SINV), the prototype virus in this genus. SINV, like the other alphaviruses, enters cells by receptor-mediated endocytosis and after fusion in the endosome releases the positive-strand RNA genome, which is translated in the cytoplasm to generate a nonstructural polyProtein, P123 (reviewed in reference 36). Translational readthrough of an opal termination codon also results in the production of the polyProtein P1234. The activity of a cysteine protease residing in nsP2 mediates polyProtein processing, which results in the generation of P123 and nonstructural Protein 4 (nsP4), the viral RNA-dependent RNA polymerase. Together, P123 and nsP4 produce a full-length negative-strand copy of the genomic RNA. Further regulated sequential processing of the polyProtein results in replication complexes active in both positive and negative strand RNA synthesis. After complete proteolytic processing, replication complexes containing the individual nsP1, nsP2, nsP3, and nsP4 Proteins are capable of only positive-strand RNA production, including both genomic RNA and a subgenomic RNA colinear with the 3′ end of the genome and encoding the viral structural Proteins. The roles of the individual viral Proteins in viral RNA replication have been investigated by examination of temperature-sensitive mutants, as well as targeted mutagenesis (reviewed in reference 20). nsP1 interacts with cellular membranes and plays a role in capping the newly generated genomic and subgenomic transcripts. In addition to its proteolytic activity, nsP2 possesses nucleotriphosphatase and helicase activities and is involved in regulation of RNA synthesis. The nsP2 Protein of SINV, as well as that of Semliki Forest virus, also has known activity in the alteration of host cellular processes, such as the shutoff of host translation and transcription (10, 12, 14, 16, 29). Mutational analysis has demonstrated a role for nsP3 in RNA synthesis, although the precise function is not understood. Several recent nsP3 interactome studies (6, 11, 17) have identified nsP3-associated cellular factors that suggest possible roles for this viral Protein in modulation of host cellular pathways. The alphavirus nsP4 is the RNA-dependent RNA polymerase with recent biochemical studies confirming and characterizing its activity (32, 38, 39). Although roles in RNA replication for the virus-encoded nonstructural Proteins (nsPs) have been elucidated (reviewed in references 20 and 36), little is known regarding how host factors may participate in the replication process. To address this, we and others have taken proteomic approaches to identify candidate host factors that may be involved in SINV replication. Using SINV expressing nsPs tagged in frame with the green fluorescent Protein (GFP), host Proteins that interact with nsP2 (1) and nsP3 (6, 11, 17) have been identified. The results from those studies allow for the generation of hypotheses as to how SINV utilizes host factors for viral replication processes. Many alphaviruses, including SINV, express P123 at levels higher than nsP4, the RNA-dependent RNA polymerase, due to an opal termination codon between nsP3 and nsP4 (35) and due to rapid degradation of nsP4 (8). We reasoned, therefore, that nsP1, nsP2, and nsP3 likely have multiple functions within the infected cell and that host Proteins previously identified as interacting with nsP2 and nsP3 may play roles in important processes that might not be directly related to replication of the viral RNA. In fact, evidence has recently been found for multiple types of nsP3-containing complexes in SINV-infected cells (17), suggesting that the functions of these viral Proteins and their interacting host Proteins may be multifaceted. We therefore undertook studies to identify host factors that interact with nsP4, the viral RNA-dependent RNA polymerase, to identify candidate host factors that might participate more directly in viral RNA replication. In the present study we isolated a Flag-tagged version of the viral RNA-dependent RNA polymerase nsP4 from infected vertebrate cells at two different times of infection, a late time (12 h) and an earlier time (6 h), and identified the interacting host Proteins by mass spectrometry. Some Proteins, e.g., Protein arginine methyltransferase 5 (PRMT5) and unc-84 homology B (UNC84), were detected in association with nsP4 only at the earlier time point. Others, e.g., adenine nucleotide translocator 1 (ANT1) and F1-ATPase, were observed only at the late time point. Some host factors, e.g., methylosome Protein 50 (MEP50), heat shock Protein 90 (HSP90), and the Ras-GTPase-activating Protein SH3-domain-binding Protein 1 (G3BP1) and G3BP2, were associated with nsP4 at both the early and late time points. The identified Proteins may play direct roles in viral RNA replication, or may be participating in host responses aimed at limiting viral replication. Two Proteins, G3BP1 and G3BP2, identified in nsP4-containing complexes were also identified in nsP2 (1)- and nsP3 (6, 11, 17)-containing complexes in prior studies. We demonstrate here a role for these Proteins in limiting SINV replication events.
-
Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication†
Journal of virology, 2010Co-Authors: Ileana M Cristea, Heather Rozjabek, Sophiya Karki, Laura L. White, Brian T. Chait, Kelly R Molloy, Charles M Rice, Michael P Rout, Margaret R. MacdonaldAbstract:Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host Proteins. Of these, 10 Proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host Proteins may be required for the multiple functions carried out by nsP4. Two related Proteins found in association with nsP4 at both times of infection, GTPase-activating Protein (SH3 domain) binding Protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
