|Vaira, Anna Maria -|
|Lim, Hyoun-Sub -|
Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 20, 2012
Publication Date: May 1, 2012
Citation: Vaira, A., Lim, H., Bauchan, G.R., Owens, R.A., Dienelt, M.M., Reinsel, M.D., Hammond, J., Natilla, A. 2012. Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue. Journal of General Virology. 93:1814-1824. Interpretive Summary: Plant viruses infect some plant species systemically, yet are unable to spread systemically in other hosts in which the virus is able to replicate in the inoculated leaf; an understanding of the virus:host interactions allowing systemic movement may allow development of means to prevent systemic infection and the resulting losses of yield and quality. Lolium latent virus (LoLV) has flexuous particles, and infects a number of forage and turfgrass species, as well as the model host Nicotiana benthamiana. LoLV is unusual in incorporating two versions of the coat protein (CP), differing in length, into virus particles. The involvement of the two CP variants in systemic movement was investigated through mutations introduced into an infectious complementary DNA clone of LoLV. Mutants in which the longer version of the CP was not produced were unable to infect plants systemically, and were unable to produce typical viral particles, unless compensating mutations appeared following localized replication in inoculated leaves. Several compensating mutations restored different variants of the larger CP of different sizes. The region of the larger CP that was restored in the mutants able to spread systemically was determined to include a chloroplast signal peptide directing the CP to the chloroplast, revealing the importance of chloroplast interactions for systemic movement of LoLV. Further mutants of the CP revealed that cleavage of the larger CP form at the chloroplast to yield the smaller form significantly delays systemic movement of LoLV. The larger CP form was also found to interact with a host protein that may aid in direction of the CP to the chloroplast. Identification of chloroplast interactions as a necessary step in systemic viral movement opens the way for possible disruption of these interactions to block systemic infection. This information will benefit scientists working on virus:host interactions, and potentially plant breeders working to introduce virus resistance.
Technical Abstract: The Lolium latent virus (LoLV, Lolavirus, Alphaflexiviridae) viral genome is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportion. The CP ORF contains two 5'-proximal AUGs, encoding Met 1 and Met 49, respectively promoting translation of the 33kDa and 28kDa CP variants. The 33kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast Transit Peptide (cTP), leading to protein cleavage. Cellular localization studies using fluorescent fusion proteins of N-terminal deletion mutants suggest that the cTP is essential for mesophyll targeting and interaction with chloroplasts. The N-terminal sequence is also crucial for efficient cell-to-cell movement, functional systemic movement, homologous coat protein interactions and particle formation, but is not required for virus replication. The 28kDa CP, but not the 33kDa CP, is associated with membranes of intact chloroplasts isolated from infected tissue and lack of production of the 28kDa CP has a dramatic effect on virus infection. Protein interaction assays demonstrate that the host ankyrin repeat protein ANK-1 is involved in LoLV CP-chloroplast targeting, the key step for most N-terminus functions.