Mutagenesis of Tyrosine 24 in the VPg P

rotein is Lethal for Feline Calicivirus

Mitra, Sosnovtsev and Green (2004)

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.

• VPg of RHDV has been recently observed to be u

ridylylated by recombinant RHDV polymerase in
dicating that calicivirus VPg may also function in
RNA replication.
VPg Protein of FCV
- 111 amino acids long
- (AA 961-1072, 12.65 kb) Two Conserved Regions
- Two amino acids motifs are c 1. K G K (N/T) K

onserved compared to other 2. (D/E) EY (D/E) E

calicivirus Vpg proteins The second one contains a tyrosine resid
ue where uridylylation occurs, and may
be the site of linkage to the viral RNA.
• Mutations of the tyrosine or serine involved in li
nkage of the RNA to the VPg protein are lethal fo
r virus growth and replication.
Picornavirus VPg protein is
uridylylated by the 3D poly
merase to form Vpg- pU and
VPg- pUpU.

VPg functions as a primer fo

r RNA synthesis during repl
ication.
Methods
• Identification of Y residues in t
he VPg region of the genome

• Construction of mutants using

site-directed mutagenesis

• 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

For the constructs containing mutations at T-24, viable viruses

could not be recovered.
Confirmation of Virus Recove
ry
 To confirm that failure of virus recovery was
not due to defective RNA synthesis:

Capped RNA transcripts

Gel analysis
RNA stained were synthesized in vitro.
with EtBr

• mRNA capping
Not-1 Linearized plasmid DNA
Capped RNA transcripts were
produced

• Arrow indicates full length

Lane 2- pQ14 parental plasmid RNA.
Lane 3- Y24A • ~5kb smaller transcript associ
Lane 4- Y24S
ated with transcription of FCV
Lane 5- Y24T
cloned genome
Lane 6- Y24F
 To confirm that failure of virus recovery
was not due to defective protein
synthesis from mutagenized plasmids:
• Capped RNA transcripts ->

• Translated into RLL

• Labeled with methionine

• Proteins were analyzed with S

DS-PAGE.

• Comparisons were made betwe

Lane 2 pQ14 parental plasmid Lane 1 is
en wild type and mutations.
previously
Lane 3 Y24A characterized
proteins from
Lane 4 Y24S TNT of FCV
Lane 5 Y24T ORF1 clone
pTMF-1 for
Lane 6 Y24F comparison
To verify that failure to recover virus was n
ot due to aberrant transcription or translati
on
• MVA/T7 infected CRFK cells w
ere transfected with wild type
and mutagenized plasmids.

• Proteins were radiolabeled.

• Cell lysates were prepared and

incubated with p-39 specific se
rum  precipitation of Ag-Ab
complexes with Sepharose pro
tein A beads.
• Analysis using SDS-PAGE and
autoradioography
Mutants and wild-type transfections showed
similar levels of mature p39 protein expression
using MVA/T7.
Thus, non-structural proteins were synthesized in
transfected cells; however, structural protein
synthesis did not occur.
Comparison of growth properties of recovered mu
tant viruses and wild-type viruses.

Growth kinetics of wild-type and mutant viruses were similar on plaque

assays except for viruses with mutations on Y24A.

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.

After infection, the virus

titer was determined by
the plaque assay.

Mutant viruses showed

similar growth kinetics to
the wild-type virus.
Examination of effects of VPg mutations on proteolytic processing of t
he ORF-1 polyprotein.
Cells infected with Y12A, Y76A
and Y104A or wild-type were
analyzed by
immunoprecipitation with
VPg- specific antibodies.

VPg precursors and 2 forms of

VPg were observed in the
wild-type and three mutations
(Y12A, Y76A and Y104A).

Thus, non-lethal mutations do

not affect proteolytic
processing of ORF1.
Conclusion
• Data in this study has shown that tyrosine 24 of t
he FCV VPg protein is essential for FCV replicati
on.