Improved third-generation lentiviral packaging with pLKO.1C vectors

A widely used third-generation lentiviral packaging system produces virus with enhanced biosafety by eliminating HIV accessory genes and separating packaging elements into three different plasmids. However, for certain vectors such as pLKO.1, third-generation safety features reduce lentiviral titers due to the lack of the accessory gene tat. Here we present a way to improve virus production and target gene knockdown with a modified pLKO.1 CMV pLKO.1C) vector and optimized packaging construct ratios. Replacing the pLKO.1 RSV promoter with the Cytomegalovirus promoter yielded an average of threefold higher titer than standard pLKO.1 packaged using the third-generation system, while optimizing the packaging vector ratios further increased titer and yielded an average of tenfold higher titer than pLKO.1 packaged with the second-generation system.

West Nile Virus Genome with Glycosylated Envelope Protein and Deletion of Alpha Helices 1, 2, and 4 in the Capsid Protein Is Noninfectious and Efficiently Secretes Subviral Particles

Flavivirus genomes with deletions in the capsid (C) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (SVPs) without generating infectious virus. Here, we report that cytomegalovirus promoter-driven cDNA of West Nile virus Kunjin (KUNV) containing a glycosylation motif in the envelope (E) gene and a combined deletion of alpha helices 1, 2, and 4 in C produces significantly more SVPs than KUNV cDNAs with nonglycosylated E and various other deletions in C.