A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions.
The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584-594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2-4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.
Rajamäki
Minna-Liisa
ML
Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland.
Kelloniemi
Jani
J
Alminaite
Agne
A
Kekarainen
Tuija
T
Rabenstein
Frank
F
Valkonen
Jari P T
JP
D000595:Amino Acid Sequence; D004251:DNA Transposable Elements; D005796:Genes / Q000235:genetics; D005818:Genetic Engineering; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D014764:Viral Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins