Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.
Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.
Casais
Rosa
R
Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Laboratorio de Sanidad Animal, Jove del Medio, Gijón, Asturias, Spain.
Molleda
Lorenzo González
LG
Machín
Angeles
A
del Barrio
Gloria
G
Manso
Alberto García
AG
Dalton
Kevin P
KP
Coto
Ana
A
Alonso
José Manuel Martín
JM
Prieto
Miguel
M
Parra
Francisco
F
D000818:Animals; D000914:Antibodies, Viral / Q000378:metabolism; D002522:Chlorocebus aethiops; D003902:Detergents / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D007668:Kidney / Q000166:cytology* / Q000821:virology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012367:RNA, Viral / Q000378:metabolism; D011817:Rabbits; D014709:Vero Cells; D029342:Vesivirus / Q000187:drug effects / Q000254:growth & development*; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000914:Antibodies, Viral; D003902:Detergents; D012367:RNA, Viral; D014764:Viral Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40