Development of infectious cDNA clones of Grapevine leafroll-associated virus 3 and analyses of the 5′ non-translated region for replication and virion formation

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3′-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5′ non-translated region (5′ NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5′ NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5′-terminus were indispensable for replication, compared to downstream variable portion of the 5′ NTR. Some of the functional mutations in the 5′ NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.