Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2

J. García-Cordero, S. Carrillo-Halfon, M. León-Juárez, H. Romero-Ramírez, P. Valenzuela-León, M. López-González, L. Santos-Argumedo, B. Gutiérrez-Castañeda, J. A. González-Y-Merchand, L. Cedillo-Barrón

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication. © 2014 Informa Healthcare USA, Inc.
Original languageAmerican English
Pages (from-to)28-40
Number of pages23
JournalImmunological Investigations
DOIs
StatePublished - 1 Jan 2014

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Dengue Virus
DNA-Directed RNA Polymerases
Monoclonal Antibodies
Virus Replication
Proteins
Methyltransferases
RNA Caps
Viral Nonstructural Proteins
Clone Cells
Western Blotting
RNA
Lamin Type A
Staining and Labeling
RNA Replicase
Antibody Specificity
Antibodies
Viral Proteins
Haplorhini
Epitopes
Delivery of Health Care

Cite this

García-Cordero, J., Carrillo-Halfon, S., León-Juárez, M., Romero-Ramírez, H., Valenzuela-León, P., López-González, M., ... Cedillo-Barrón, L. (2014). Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2. Immunological Investigations, 28-40. https://doi.org/10.3109/08820139.2013.833622
García-Cordero, J. ; Carrillo-Halfon, S. ; León-Juárez, M. ; Romero-Ramírez, H. ; Valenzuela-León, P. ; López-González, M. ; Santos-Argumedo, L. ; Gutiérrez-Castañeda, B. ; González-Y-Merchand, J. A. ; Cedillo-Barrón, L. / Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2. In: Immunological Investigations. 2014 ; pp. 28-40.
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abstract = "Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication. {\circledC} 2014 Informa Healthcare USA, Inc.",
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García-Cordero, J, Carrillo-Halfon, S, León-Juárez, M, Romero-Ramírez, H, Valenzuela-León, P, López-González, M, Santos-Argumedo, L, Gutiérrez-Castañeda, B, González-Y-Merchand, JA & Cedillo-Barrón, L 2014, 'Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2', Immunological Investigations, pp. 28-40. https://doi.org/10.3109/08820139.2013.833622

Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2. / García-Cordero, J.; Carrillo-Halfon, S.; León-Juárez, M.; Romero-Ramírez, H.; Valenzuela-León, P.; López-González, M.; Santos-Argumedo, L.; Gutiérrez-Castañeda, B.; González-Y-Merchand, J. A.; Cedillo-Barrón, L.

In: Immunological Investigations, 01.01.2014, p. 28-40.

Research output: Contribution to journalArticle

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T1 - Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2

AU - García-Cordero, J.

AU - Carrillo-Halfon, S.

AU - León-Juárez, M.

AU - Romero-Ramírez, H.

AU - Valenzuela-León, P.

AU - López-González, M.

AU - Santos-Argumedo, L.

AU - Gutiérrez-Castañeda, B.

AU - González-Y-Merchand, J. A.

AU - Cedillo-Barrón, L.

PY - 2014/1/1

Y1 - 2014/1/1

N2 - Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication. © 2014 Informa Healthcare USA, Inc.

AB - Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication. © 2014 Informa Healthcare USA, Inc.

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García-Cordero J, Carrillo-Halfon S, León-Juárez M, Romero-Ramírez H, Valenzuela-León P, López-González M et al. Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2. Immunological Investigations. 2014 Jan 1;28-40. https://doi.org/10.3109/08820139.2013.833622