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Journal of Virology, September 2002, p. 8582-8595, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8582-8595.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells

Kim Y. Green,1* Aaron Mory,2 Mark H. Fogg,1 Andrea Weisberg,1 Gaël Belliot,1 Mariam Wagner,1 Tanaji Mitra,1 Ellie Ehrenfeld,1 Craig E. Cameron,2 and Stanislav V. Sosnovtsev1

National Institutes of Health, Bethesda, Maryland,1 Pennsylvania State University, University Park, Pennsylvania2

Received 15 February 2002/ Accepted 6 June 2002

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.

* Corresponding author. Mailing address: National Institutes of Health, Building 50, Room 6318, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 594-1665. Fax: (301) 480-5031. E-mail: kgreen{at}niaid.nih.gov.

Journal of Virology, September 2002, p. 8582-8595, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8582-8595.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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