Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments.
ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.
Department of Virology, Biological Faculty, Lomonosov Moscow State University, Moscow 119991, Russia.|Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.|The James Hutton Institute, Dundee DD2 5DA, Scotland, UK.
Vladimir A
VA
Gushchin
Department of Plant Biology and Forest Genetics, Uppsala BioCentre, Swedish University of Agricultural Sciences, Uppsala 75007, Sweden.
Nina I
NI
Lukhovitskaya
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
Dmitri E
DE
Andreev
The James Hutton Institute, Dundee DD2 5DA, Scotland, UK.
Kathryn M
KM
Wright
The James Hutton Institute, Dundee DD2 5DA, Scotland, UK.
Michael E
ME
Taliansky
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.|Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow 127550, Russia.
Andrey G
AG
Solovyev
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
Sergey Y
SY
Morozov
The James Hutton Institute, Dundee DD2 5DA, Scotland, UK.
Stuart A
SA
MacFarlane
D002467:Cell Nucleus / Q000821:virology*; D004721:Endoplasmic Reticulum / Q000821:virology*; D008928:Mitochondria / Q000821:virology*; D009154:Mutation; D016366:Open Reading Frames; D010942:Plant Viruses / Q000235:genetics / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D014764:Viral Proteins