Moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response

Vesicular stomatitis vines (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome en codes five genes in the order 3'-N-P-M-G-L-5', and transcription is obligat orily sequential from the single 3' promoter. As a result, genes at promote r-proximal positions are transcribed at higher levels than those at promote r-distal positions, Previous work demonstrated that moving the gene encodin g the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachme nt glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expr ession in cells and alter the immune response in inoculated animals. In add ition to moving the G gene alone, we also constructed viruses having both t he G and N genes rearranged. This produced three variant viruses having the orders 3'-G-N-P-M-L-5' (G1N2), 3'-P-M-G-N-L-5' (G3N4), and 3'-G-P-M-N-L-5' (G1N4), respectively, These viruses differed from one another and from wil d-type virus in their levels of gene expression and replication in cell cul ture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge,vith wild-type VSV. Transloca tion of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replica tion and lethality for animals. These studies demonstrate that gene rearran gement can be exploited to design nonsegmented negative-sense RNA viruses t hat have characteristics desirable in candidates for live attenuated vaccin es.