Study Of Dengue Virus Capsid Targeted Viral Inactivation

Dengue virus, a member of the flavivirus genus of enveloped RNA virus, is one of the most significant viral pathogens and now presents in over 100 countries. Annually, the four serotypes of dengue virus collectively cause 100 million or more cases of infection. Dengue infection is usually characterized by fever and joint pains, but more serious syndromes, dengue hemorrhagic fever and dengue shock syndrome(combined mortality, up to 5%), sometimes occur following infection. Although intense efforts have been made to control those diseases, dengue is still endemic and epidemic and threatens the health of over 2.5 billion people in urban and rural areas of the tropics and subtropics. Dengue infection has now been a major global public health problem.Currently, no vaccine or effective antiviral treatment exists for the prevention or treatment of infections with dengue virus. This situation has spurred urgently the interest in search for new strategy against dengue. One potential approach, termed intracellular inhibition or immunization, was designed to render cells resistant to viral pathogens by introducing genes encoding antiviral macromolecules into a cell culture or an individual. Inhibition of dengue viral multiplication by such a strategy had been established with different antiviral genes, most of which were nucleic-acid-based. Capsid targeted viral inactivation (CTVI), first proposed by Natsoulis and Boeke, represented a conceptually powerful protein-based antiviral approach. In this strategy the viral capsid protein was designed as carrier to target a deleterious enzyme, e.g., a nuclease, a proteinase and even a single chain antibody bind to a native viral protein, specifically into progeny virions during assembly to prevent the production of infectious viral particles and subsequent spread of de novo infection. CTVI had beenthoroughly investigated in the experimental treatments for several important virus, such as murine leukemia virus, hepatitis B virus and human immunodeficiency virus, showing a promising prospect as an antiviral strategy.In this paper, we firstly cloned and expressed the dengue 2 virus capsid protein, termed with D2C, and staphylococcal nuclease(SN) genes respectively with E.coli GI724/pLEX system from Invitrogen. The recombinant expressed D2C was about 11kDa and could be recognized by monoclonal antibody 8H8 raised against dengue 2 virus A15 strains capsid protein. Unexpectedly, there were no evidence showing the ability of self-assembly into capsid-like particles in vitro of recombinant D2C protein. The bacterial expressed SN was immuned rabbits to prepare polyantibody against SN for further detection of eukaryotic expression, and the prepared antisera were specfic to recognize recombinant SN and SN*.To explore the feasibility of CTVI as an antiviral strategy against dengue infection, first of all we fused SN gene to D2C gene at the carboxyl terminus, to construct the antiviral effector D2C-SN. This orientation was chosen so that the nuclease could be inserted within the particles where it would have access to the viral RNA. The E.coli. expressed D2C-SN was about 30kDa and could be recognized by Mab 8H8. What's more, the recombinant expressed D2C-SN had proper nuclease activity to degrade DNA or RNA with appropriate Ca2+ concentrations. Those data supported the further research.Based on the previous work, we inverted the constructed D2C-SN and D2C-SN* gene fragments with EcoR I and Xho I sites into the eukaryotic expression vector pcDNA6 to obtain the desired recombinant palsmids pc/D2C-SN and pc/D2C-SN*. Firstly, we examined the temporary expression of D2C-SN in mammalian BHK21 cells. After transfection by electroporation, RT-PCR? Western Blot and indirect immunofluorescence were used to detect the expression of D2C-SN. The results indicated that D2C-SN could be expressed and detected with specific antibody against D2C and SN. And the results of MTT assay showed that D2C-SN were no cytotoxic to the host cell. Secondly, we set up prophylactic and therapeutic models to demonstrat...