Production of both carboxy-coterminal coat protein forms of Lolium latent virus is required for efficient systemic movement

The Lolium latent virus (LoLV, Lolavirus, Alphaflexiviridae) genome is encapsidated by equimolar amounts of carboxy-coterminal coat protein (CP) variants of apparent MW 33 and 28 kDa. The CP ORF contains two 5´-proximal AUGs, encoding Met 1 and Met 49, respectively promoting translation of the 33 kDa and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast Transit Peptide (cTP) with a predicted cleavage site upstream of AUG2. Ablation of AUG1 in an infectious clone yielded mutant LoLV-K1, which was able to replicate in inoculated leaves of Nicotiana benthamiana, but not spread systemically. Mutation of AUG2 to UUG yielded mutant LoLV-K2, which was able to infect plants systemically. LoLV-K1 revertants that regained expression of a CP form of >28 kDa (by restoration of wild-type AUG1; by mutation to AUG at another site; or by mutation to an upstream CUG alternate initiation codon) were able to infect plants systemically. Substitution of four amino acids at the predicted cTP cleavage site combined with AUG2>CCC yielded mutant LoLV-C4, in which systemic infection was significantly delayed and symptoms altered. The N-terminal cTP sequence is crucial for efficient cell-to-cell and systemic movement, as well as homologous CP interactions and particle formation, but is not required for virus replication. Lack of production of the 28 kDa CP by either internal initiation or proteolytic cleavage limits systemic infection.