The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5'-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloroplast membrane has a dramatic effect on virus infection.