Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55Gag that is capable of producing virus-like particles

Ectopic expression of the structural protein Pr55Gag of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5′-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3′-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55Gag in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55Gag derived from this JEV replicon RNA appeared to be increased by ∼3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55Gag proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55Gag could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55Gag by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.