|Morris, T - UNIVERSITY OF NEBRASKA|
Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 9, 2000
Publication Date: January 8, 2000
Citation: Robertson, N.L., French, R.C., Morris, T.J. 2000. The open reading frame 5a of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection. Archives Of Virology 145:1685-1698. Interpretive Summary: Foxtail mosaic potexvirus (FoMV) infects many cereal crop plants and may be useful for a variety of biotechnology applications such as a gene expressio vector. The complete genome of FoMV was cloned in a plasmid. The resultant vitro RNA transcripts were infectious to barley and Chenopodium amaranticolor. The infectious clones were then used for an initial characterization of virus gene function. Individual mutation studies on clones of each of the five major genes confirmed predicted functions for th polymerase, triple gene block movement proteins, and coat protein genes. Protoplast studies on expression of a unique open reading frame, ORF 5A, which begins in front of the coat protein, revealed that the 5A protein was produced during infection. Mutations were made in the 5A protein gene which demonstrated that it is not required for replication or for productive infection of plants. However, the nucleic acid sequences encoding this gene esegment were shown to be important for coat protein production. These mutations had no effect on FoMV replication in protoplasts but rendered the virus noninfectious to plants. This implies that the amount of coat protei made must exceed a minimal value for infection to occur.
Technical Abstract: Infectious transcripts were successfully derived from full-length cDNA clon of foxtail mosaic potexvirus (FoMV). Full-length clones were constructed by RT-PCR whereby 5 and 3 genomic segments of 2.7 and 3.4 kbp, respectfully, were ligated into Bluescript II KS phagemid. The resultant in vitro RNA transcripts were infectious to moncotyledonous (barley) and dicotyledonous (Chenopodium amaranticolor) plant species. Individual mutation studies on clones of each of the five major ORFs confirmed predicted gene function for the polymerase, TGB (triple gene block), and coat protein (cp) genes. Protoplast studies on expression of a unique open reading frame, ORF 5A, which initiates 143 nts upstream of the coat protein before it "reads through" the cp, revealed that the 5A protein was produced in vivo. Further mutagenesis of the 5A ORF indicated that it was not required for either replication or for productive infection of plants. However, the nucleic aci isequences encoding the extended cp segment were shown to be important for subgenomic mRNA production. This was established by a point mutation and an in-frame addition of six nucleotides within the unique 5A region. These mutations had no effect on FoMV replication in protoplasts but rendered the virus noninfectious to plants. A correlation with diminished cp production from both mutant clones implies that the cp synthesis of subgenomic RNA was compromised, and this limited systemic infection.