Flaviviruses are positive, single-stranded, enveloped cytoplasmic sense RNA viruses that cause a variety of important diseases worldwide. Among them, Zika virus, West Nile virus, Japanese encephalitis virus, and Dengue virus have the potential to cause severe disease. Extensive studies have been performed to elucidate the structure and replication strategies of flaviviruses, and current studies are aiming to unravel the complex molecular interactions between the virus and host during the very early stages of infection. The outcomes of viral infection and rapid establishment of the antiviral state, depends on viral detection by pathogen recognition receptors and rapid initiation of signalling cascades to induce an effective innate immune response. Extracellular and intracellular pathogen recognition receptors play a crucial role in detecting flavivirus infection and inducing a robust antiviral response. One of the main hallmarks of flaviviral nonstructural proteins is their multiple strategies to antagonise the interferon system. In this chapter, we summarize the molecular characteristics of flaviviral proteins and discuss how viral proteins target different components of the interferon signalling pathway by blocking phosphorylation, enhancing degradation, and downregulating the expression of major components of the Janus kinase/signal transducer and activator of transcription pathway. We also discuss how the interactions of viral proteins with host proteins facilitate viral pathogenesis. Due to the lack of antivirals or prophylactic treatments for many flaviviral infections, it is necessary to fully elucidate how these viruses disrupt cellular processes to influence pathogenesis and disease outcomes.
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.
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