- Papain-like leader proteases are found in diverse families of human, animal, plant, and fungal positive-strand RNA viruses. In addition to autocatalytic processing, these proteases play a variety of roles in the virus life cycle. In particular, the leader protease (L-Pro) of a prototype member of the Closterovirus genus, Beet yellows virus (BYV), was implicated in autoproteolytic processing, genome amplification, and long-distance transport. The genetic organization of Grapevine leafroll-associated virus-2 (GLRaV-2) is similar to that of BYV, however, GLRaV-2 codes for two leader proteases, L1 and L2. Previous work suggested that the tandems of leader proteases in GLRaV-2 and other closteroviruses emerged via independent gene duplication events. It was also proposed that the evolution of L1 and L2 involved functional divergence (neofunctionalization) that resulted in the erosion of sequence similarity in the N-terminal non-proteolytic domains. This study was designed to characterize the functional profiles of GLRaV-2 leader proteinases.
Because the infectious cDNA clones of the RNA viruses are indispensable for investigation of viral gene functions, we generated such cDNA clone for GLRaV-2. To facilitate observation and quantification of each phase of the virus infection cycle, cDNA clones were tagged via insertion of fluorescent, enzymatic, and epitope reporters. The tagged clones were used to study the functions of GLRaV-2 L1 and L2. We found that the autoproteolytic processing by L2 but not by L1 is critical for virus viability. It was also revealed that L1 and L2 have complementary and overlapping functions in the establishment of the viral infection in the initially inoculated cells, and, to different extent, in the systemic transport of GLRaV-2. Strikingly, we have demonstrated that the overall contributions of L1 and L2 into virus infection are much more critical in a natural virus host, grapevine, compared to an experimental herbaceous host Nicotiana benthamiana, suggesting that the tandem of leader proteases evolved to facilitate an expansion of the Closterovirus host range into woody plants.