Development of a Minireplicon for Grapevine Leafroll-Associated Virus 1 and Genetic Analyses of Sequences in the 5ʹ Non-Translated Region Required for Replication

Grapevine leafroll-associated virus 1 (GLRaV-1, genus <i>Ampelovirus</i>, family <i>Closteroviridae</i>) has a monopartite RNA genome with the size varying among genetic variants between 18,731 and 18,946 nucleotides (nt) and a 5' non-translated region (5'-NTR), varying in length between 857 and 922 nt. This study was undertaken to examine the role of the 5'-NTR in GLRaV-1 replication. For this purpose, a minireplicon cDNA clone, consisting of the 5'-NTR, the replicase gene module, the green fluorescent protein (GFP) reporter gene, and the 3'-NTR, was constructed. The functionality of the minireplicon was validated by GFP fluorescence and the presence of GFP-specific mRNA transcripts by Northern blot hybridization and reverse transcription quantitative PCR assays in <i>Nicotiana benthamiana</i> leaves agro-coinfiltrated with silencing suppressors. The minireplicon retained functionality when its 5'-NTR was exchanged with corresponding sequences from distinct genetic variants of GLRaV-1. In contrast, the minireplicon of GLRaV-1 was nonfunctional when its 5'-NTR sequence was swapped with corresponding sequences from other GLRaV species. Deletion mutations in the 5'-NTR indicated that the first 32 nt at the 5'-terminus of the genome are essential for replication of the minireplicon. The in silico-predicted secondary structure of the 5'-terminal 32-nt sequence showed two stem-loop structures and mutations that altered these secondary structures or compensatory mutations preserving the secondary structures that failed to retain functionality of the minireplicon, suggesting that the nucleotide sequence, rather than any higher order secondary structures in this genomic region, is important for replication.