Construction and Characterization of Feline Immunodeficiency Virus Proviral Mutants That Coexpress Interferon Gamma and Green Fluorescent Protein

Abstract

Vif deletion mutants of the feline immunodeficiency virus (FIV) were designed to express either enhanced green fluorescent protein (EGFP) (FIVΔvifATGgfp) or feline interferon-γ (IFN-γ) (FIVΔvifATGγ) by insertion of the nonviral gene into the deletion site of the viral vif gene. Two in-frame start codons within vif were mutated without altering the overlapping pol translation frame to enhance expression of inserted genes. Expression of EGFP and IFN-γ from FIVΔvifATGgfp and FIVΔvifATGγ proviruses, respectively, was confirmed by fluorescent microscopy and immunocytochemical assays, respectively. Replication of viruses generated from these proviruses was detectable but severely restricted when compared to that of wild-type (WT) FIV-pPPR. A previous study demonstrated induction of protection against homologous FIV challenge by vaccination of cats with an attenuated FIV-pPPRΔvif proviral DNA vaccine (Lockridge K et al.: Virology 2000;273:67–79). Coexpression of IFN-γ or other cytokines from this attenuated provirus provides the opportunity to evaluate the ability of an immunomodulator to enhance the safety and efficacy of an infectious attenuated DNA vaccine. Moreover, a vif-deleted FIV provirus that coexpresses a reporter gene such as EGFP may be used to examine the localization of vif mutant viruses in vivo.