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

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.

Competitive suppression of dengue virus replication occurs in chikungunya and dengue co-infected Mexican infants

“…Once in suspension, cells were treated with fixing/permeabilising solution (Becton Dickinson, New Jersey, USA) at 4 °C for 1 h with gentle agitation, washed twice with PBS/0.5% albumin, and treated for 30 min with an unrelated immunoglobulin solution (10% goat serum in PBS). Cells were then incubated with rat anti-NS5 monoclonal antibody for DENV [ 18 ] and with a mouse polyclonal anti E2 for CHIKV. After 30 min incubation, the cells were treated with the corresponding secondary antibodies: anti-mouse IgG H+L CY3 and anti-rat-FITC IgG H+L.…”

“…Briefly, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 20 min at room temperature, permeabilized with a solution of PBS supplemented with 0.1% Triton-X100 and blocked with 10% normal goat serum. The cell monolayer was incubated for 60 min with primary antibodies as follows: mAb 4G2 for detection of DENV-1, anti-NS5 for DENV-2 or anti-E2 protein for CHIKV [ 18 ]. After washing, the following fluorochrome-conjugated secondary antibodies were added: mouse IgG H+L for DENV-1 (Invitrogen, Carlsbad, CA, USA) or rat IgG H+L for DENV-2 and CHIKV (Invitrogen, Carlsbad, CA, USA).…”

Caveolin-1 in Lipid Rafts Interacts with Dengue Virus NS3 during Polyprotein Processing and Replication in HMEC-1 Cells

“…The mouse monoclonal antibody directed against NS3 (D-7) and the rat monoclonal antibody against NS5 (13G7) have been previously described [44], [45].Rabbit polyclonal anti-Cav-1 and mouse monoclonal anti-Flt-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rat polyclonal antibodies targeting NS2B and NS3 were produced in our laboratory.…”

Letter from the Editor

“…A pandemic H1N1 influenza virus-like particle vaccine induces crossprotection in mice (Inn et al, 2014) Dicer in immune cell development and function (Devasthanam & Tomasi, 2014) Antinuclear antibodies: two-step detection strategy (Callado et al, 2014) Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2 (García- Cordero et al, 2014) Study of TLR4 and IL-8 Gene Polymorphisms in H. pylori-Induced Inflammation in Gastric Cancer in an Ethnic Kashmiri Population (Qadri et al, 2014). TGF-b favors bone marrow-derived dendritic cells to acquire tolerogenic properties (Song et al, 2014).…”