Biochemical characterization of the helper component of Cauliflower mosaic virus.
The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.
Hebrard
E
E
Station de Recherches de Pathologie Comparée, UMR 5087, INRA-CNRS-Université Montpellier II, 30380 Saint-Christol-les-Alès, France.
Drucker
M
M
Leclerc
D
D
Hohn
T
T
Uzest
M
M
Froissart
R
R
Strub
J M
JM
Sanglier
S
S
van Dorsselaer
A
A
Padilla
A
A
Labesse
G
G
Blanc
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017796:Caulimovirus / Q000737:chemistry*; D002460:Cell Line; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D009842:Oligopeptides; D011108:Polymers; D014764:Viral Proteins