Immunolocalization of Picornavirus RNA in infected cells with antibodies to Tyr-pUp, the covalent linkage unit between VPg and RNA.
The genomic RNA of picornaviruses is attached to a small protein (VPg) via a covalent bond between a tyrosine and a 5'-terminal uridine phosphate. The same structure is present in potyvirus and calicivirus families. VPgs play a key role in initiation of viral replication by acting as primers for RNA synthesis. The model compound [N(Ac),CO(NHMe)]Tyr-(5'P→O)Up-O-(CH(2))(6)NH(2) (mCLU), mimicking this 'covalent linkage unit' (CLU) and containing Tyr-pUp was synthesized in solution following the phosphoramidite scheme and used to raise antibodies for studying picornavirus infection. The antibodies recognized CLU-containing mengovirus RNA and showed minimal cross-reactivity with RNAs lacking CLU. Immunofluorescence staining of cells infected with a human rhinovirus demonstrated co-localization of the signals from anti-mCLU and from anti-VPg antibodies. Efficient synthesis of mCLU and anti-mCLU antibodies might be of great utility for investigating viral replication and identifying yet unknown viral and cellular CLU-containing RNA-protein complexes.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia. esgavryushina83@mail.ru
Elena S
ES
Gavryushina
Sofia A
SA
Bryantseva
Elena S
ES
Nadezhdina
Timofei S
TS
Zatsepin
Ilya Yu
IY
Toropygin
Angela
A
Pickl-Herk
Dieter
D
Blaas
Yuri F
YF
Drygin
D000818:Animals; D000914:Antibodies, Viral / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000276:immunology*; D010849:Picornaviridae / Q000737:chemistry / Q000254:growth & development*; D012367:RNA, Viral / Q000032:analysis*; D011817:Rabbits; D014773:Virology / Q000379:methods*
D000914:Antibodies, Viral; D009843:Oligoribonucleotides; D012367:RNA, Viral; C040065:VPg-pUpU