TY - JOUR
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.
N1 - Funding Information:
This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT, México) Grant CB2010-01-154270 and salud-2011-01-115401. Additionally, SCH, MLJ, PVL and MGL received predoctoral fellowships from CONACyT.
PY - 2014
Y1 - 2014
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.
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.
KW - DENV NS5
KW - Dengue virus
KW - Monoclonal antibody
UR - http://www.scopus.com/inward/record.url?scp=84889860901&partnerID=8YFLogxK
U2 - 10.3109/08820139.2013.833622
DO - 10.3109/08820139.2013.833622
M3 - Artículo
SN - 0882-0139
VL - 43
SP - 28
EP - 40
JO - Immunological Investigations
JF - Immunological Investigations
IS - 1
ER -