Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2...

Autonomously replicating Bovine viral diarrhea virus (BVDV) genomes (replicons) were constructed from the full-length BVDV cDNA clone pA/BVDV/Ins- (G. Meyers et al., J. Virol. 70, 8606-8613, 1996). The sequences coding for envelope protein E2, for E2 without the C-terminal transmembrane region, or for E2 and nonstructural protein p7 were deleted, and the resulting mutants were tested for their ability to replicate after transfection. All deletion mutants were able to replicate and to express the inserted green fluorescent protein but did not produce infectious progeny virus in bovine kidney PT cells. The replicons were also tested for their ability to be trans-complemented in the bovine cell line PT_805, which constitutively expresses BVDV structural proteins. E2-negative BVDV mutants were complemented and >10(6) infectious units were obtained at 24 h after transfection. Complementing PT_805 cells could only inefficiently be infected using trans-complemented virions, however, and low levels of virus production were observed when complemented BVDV was passaged using PT_805 cells. Similarly, infection of PT_805 cells with BVDV was highly inefficient, but transfection of full-length BVDV NCP7 RNA into PT_805 resulted in 10,000-fold higher virus titers when compared to those obtained 24 h after transfection of parental PT cells. We concluded that self-replicating E2-deleted BVDV RNAs can be efficiently trans-complemented by constitutively expressed E2, and that expression of BVDV structural proteins markedly influences susceptibility of cells to BVDV infection as well as BVDV titers after transfection of full-length BVDV RNA.