Insertion of cellular NEDD8 coding sequences in a pestivirus

For the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP 82 1, a duplication of the genomic region encoding part of NS2, NS3, NS4A, and part of NS4B together with a nonviral insertion was detected. Further anal yses including molecular cloning and sequencing of the putative cellular re combination partner showed that the insertion in CP 821 originated from a b ovine mRNA encoding the cellular protein NEDD8, which is 58% identical to u biquitin. To our knowledge the genome of CP 821 represents the first viral RNA with a NEDD8 coding insertion. Remarkably, the insertion site differs f rom that described for insertions of ubiquitin. The NEDD8 sequence allows a n additional cleavage of the viral polyprotein, whereby an NS3 with an unus ual N-terminus is generated. Furthermore, the CP 821-specific genomic alter ations were introduced into an infectious noncytopathogenic (noncp) BVDV cD NA clone. After transfection of bovine cells with the respective RNA, a cp virus was recovered. This showed that the NEDD8 coding insertion together w ith the duplicated viral sequences represents the genetic basis for cytopat hogenicity of CP 821. In addition to the recovered cp virus, noncp BVDV rap idly evolved after transfection. This is the first time that a change from the cp to the noncp phenotype was demonstrated in the course of replication in tissue culture cells. (C) 2000 Academic Press.