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The Lolium latent virus (LoLV) major coat protein sequence contains a typical chloroplast transit peptide (cTP) domain. In infected Nicotiana benthamiana leaf tissue, LoLV coat proteins can be detected at the chloroplast. In transient expression, several N-terminal deletions of the CP sequence, increasing in length, result in disruption of the domain functionality, markedly affecting intracellular localization. A yeast two-hybrid-based study using LoLV CP as bait identified several potentially interacting Arabidopsis host proteins, most of them with chloroplast-linked pathways. One of them, an ankyrin repeat protein, was studied in detail. The N. benthamiana homologue (NbANKr) targets chloroplasts, is able to co-localize with LoLV CP at chloroplast membranes in transient expression and shows a robust interaction with LoLV CP in vivo by BiFC, which has been confirmed by yeast two-hybrid data. Silencing NbANKr genes in N. benthamiana plants, prior to challenging with LoLV by mechanical inoculation, affects LoLV infection, significantly reducing the level of viral RNA in young leaves, compared to levels in control plants, and suggesting an inhibition of virus movement. Silencing of NbANKr has no obvious effect on plant phenotype, but is able to interfere with LoLV infection, opening the way for a new strategy for virus infection control.

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CPSP) with that from AltMV-Po (CP(Po)) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP(Po) [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CPSP but not CP(Po) interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CPSP than CP(Po) in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN., (Published by Elsevier Inc.)

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.