Isolation of enzymatically active replication complexes from feline calicivirus-infected cells.
A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.
Green
Kim Y
KY
National Institutes of Health, Bethesda, Maryland 20892, USA. kgreen@niaid.nih.gov
Mory
Aaron
A
Fogg
Mark H
MH
Weisberg
Andrea
A
Belliot
Gaël
G
Wagner
Mariam
M
Mitra
Tanaji
T
Ehrenfeld
Ellie
E
Cameron
Craig E
CE
Sosnovtsev
Stanislav V
SV
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D017250:Caliciviridae Infections / Q000821:virology; D017927:Calicivirus, Feline / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D002415:Cats; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014779:Virus Replication
D000998:Antiviral Agents; D008565:Membrane Proteins; D011498:Protein Precursors; D012367:RNA, Viral; D014764:Viral Proteins; D020126:Brefeldin A