Analysis Of Duck Viperin
Objective 1: Develop a cell line expressing the duck viperin.
In order to develop an avian model that is able to express the duck viperin with an advantage of stable and homogeneous expression, duck viperin with and without the C-terminal V5 tag was previously cloned into the mammalian expression vector pcDNA3.1/Hygro+. Then, with the use of lipofectamine 2000, I stably transfected an empty vector and duck viperin tagged or untagged into DF-1 cells. In order to get a monoclonal stable cell line, I plated on 96 well plates for isolation and separation of a single clones. To confirm the transfection, a western blot technique was used to confirm the expression of the tagged viperin in DF-1 cells; a band size with 42 kDa should be detected. …show more content…
In IAV infection of primary mouse macrophages, viperin prevented prenylation of cell membrane proteins by interacting with the enzyme FPPS and thus inhibited influenza budding from the plasma membrane, which is reversed in the presence of farnesol (Wang et al. 2007). During HIV infection of monocyte-derived macrophages (MDMs), viperin is produced and exert its inhibitory effect on HIV via inhibiting FPPS. The addition of exogenous farnesol prevented lipid raft disruption and permitted viral budding which significantly enhances HIV production in the cells (Nasr et al. 2012). In order to investigate whether duck viperin inhibits the IAV egress from the cells by inhibiting FPPS and lipid raft formation, exogenous farnesol will be added to the DF-1 cells stably expressing viperin and the percentage of infected cells will be determined using the Operetta High Content Imaging System (Perkin Elmer) and compared to DF-1 cells expressing DF-1 stably expressing viperin. I expect to see an increase in the number of infected cells with the addition of …show more content…
This experiment is still under optimization, I keep losing cells after infection and this might be due to the addition of TPCK-trypsin (L-(tosylamido-2-phenyl) ethyl chloromethyl ketone). TPCK-trypsin helps the virus to infect the cell through cleaving the HA protein into HA1 and HA2. According to Lee et al the optimum TPCK-trypsin concentration that can be used to infect DF-1 cells with IAV is 0.1 mg/ml (Lee et al. 2008). I am planning to try different concentrations to see which one will work better.
I am planning to try different concentrations of TPCK-trypsin to see which concentration will work better in order not to lose cells as well as helping the IAV to infect them. Once I get my optimal concentration that will help IAV to infect the cells as well as will decrease their loss, I will evaluate the antiviral effect of duck viperin against IAV, DF-1 cells stably expressing viperin or transfected with an empty vector will be infected with A/chicken/California/431/00 (H6N2), A/duck/Memphis/546/1974 (H11N9) and A/Puerto Rico/8/1934 (H1N1) at different time points and the percentage of infected cells will be determined using the Operetta High Content Imaging System (Perkin Elmer) or by flow cytometry