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Mutagenesis of Tyrosine 24 in The VPG Protein
Mutagenesis of Tyrosine 24 in The VPG Protein
Prepared by:
Therese Collantes, DVM
Laboratory of Animal Diseases
Department of Molecular Medicine
College of Veterinary Medicine
Chonnam National University
Introduction
• Feline Calicivirus (FCV)
▫ Member of genus Vesivirus in
the family Caliciviridae
▫ Causes respiratory illness in c
ats
• Genome
▫ ~7.7 kb
▫ Single stranded positive-sens
e RNA
▫ VPg covalently- linked at 5’ en • Three ORFs
d ▫ ORF1- 200 kDa polyprotein
▫ Polyadenylated at 3’ ▫ ORF2- 73 kDa (capsid protei
ns)
▫ ORF3- 12 kDA protein (VP2)
• VPg may play an important role in interaction wi
th cellular machinery to initiate translation.
• Verification of sequence
Recovery of viruses
• Transformation into E. coli
• Transfection into CRFK cells
• Monitoring FCV recovery by passage of cell
culture
• Cytopathic effects of FCV were observed from mutations on T1
2, T76 and T104 of the VPg.
• No cytopathic effects from the Y24A construct
• Immunofluorescence assay showed negative capsid expression
for:
▫ Original transfection
▫ Subsequent passages
(Data not shown for both cell passage and IFA)
Site- Directed Mutagenesis
• mRNA capping
Not-1 Linearized plasmid DNA
Capped RNA transcripts were
produced
CRFK cells infected with serial dilutions of viruses seeded in 6-well plates
incubated for 1 hour at 37°C cells were washed and added with agarose
overlay cells incubated at 37°C for 24 hours in humidified CO2 incubator.
Monolayers fixed with formalin and stained with crystal violet.
Growth characteristics were analyzed by measuring log virus titers.