Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.
Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or DsRedFP were recovered. The effects of the insertions on LC processing, RNA replication, and stability of the viral genome were analyzed, and the progression of a calicivirus single infection and co-infection were captured by real-time imaging fluorescent microscopy. The ability to engineer viable recombinant caliciviruses expressing foreign markers enables new approaches to investigate virus and host cell interactions, as well as studies of viral recombination, one of the driving forces of calicivirus evolution.
Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA.
Eugenio J
EJ
Abente
Stanislav V
SV
Sosnovtsev
Karin
K
Bok
Kim Y
KY
Green
D000818:Animals; D001483:Base Sequence; D017927:Calicivirus, Feline / Q000235:genetics* / Q000502:physiology*; D036022:Capsid Proteins / Q000235:genetics; D002415:Cats; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D054884:Host-Pathogen Interactions; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D014779:Virus Replication; D000097573:Red Fluorescent Protein
D036022:Capsid Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012313:RNA; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C039919:RNA, recombinant