Stabilization of cauliflower mosaic virus P3 tetramer by covalent linkage.
Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.
Tsuge
S
S
Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto Prefectural University, Kyoto, Japan. s_tsuge@love.kpu.ac.jp
Okuno
T
T
Furusawa
I
I
Kubo
Y
Y
Horino
O
O
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000821:virology; D001483:Base Sequence; D001937:Brassica / Q000821:virology; D017796:Caulimovirus / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology; D019065:Virus Assembly; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; C115486:p15 protein, Cauliflower mosaic virus; D003545:Cysteine; D000409:Alanine