mutagenesis of tyrosine 24 in the vpg protein

8/8/2019 Mutagenesis of Tyrosine 24 in the VPg Protein

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Mutagenesis of Tyrosine 24 in the

VPg Protein is Lethal for FelineCalicivirusMitra, Sosnovtsev and Green (2004)

Prepared by:Therese Collantes, DVMLaboratory of Animal DiseasesDepartment of Molecular MedicineCollege of Veterinary MedicineChonnam National University


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Introduction

Feline Calicivirus (FCV)

Member of genus Vesivirus in

the familyCaliciviridae Causes respiratory illness in

cats

Genome

~7.7 kb

Single stranded positive-sense RNA

VPg covalently- linked at 5end

Polyadenylated at 3

Three ORFs

ORF1- 200 kDa polyprotein

ORF2- 73 kDa (capsidproteins)

ORF3- 12 kDA protein (VP2)


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VPg may play an important role in interaction

with cellular machinery to initiate translation.

VPg of RHDV has been recently observed to beuridylylated by recombinant RHDV polymerase

indicating that calicivirus VPg may also functionin RNA replication.


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Two ConservedRegions1. K G K (N/T) K

2. (D/E) EY (D/E) EThe second one contains a tyrosineresidue where uridylylation occurs, andmay be the site of linkage to the viralRNA.

VPg Protein ofFCV- 111 amino acids long

- (AA 961-1072, 12.65 kb)

- Two amino acids motifs are

conserved compared toother calicivirus Vpgproteins


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Mutations of the tyrosine or serine involved in

linkage of the RNA to the VPg protein are lethalfor virus growth and replication.


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Picornavirus VPg protein isuridylylated by the 3Dpolymerase to form Vpg- pU

and VPg- pUpU.

VPg functions as a primerfor RNA synthesis duringreplication.


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Methods

Identification of Y residues inthe VPg region of the genome

Construction of mutants usingsite-directed mutagenesis

Verification of sequence


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Recovery of viruses

Transformation intoE. coli Transfection into CRFK cells

Monitoring FCV recovery by passage of cellculture Cytopathic effects ofFCV were observed from

mutations on T12, 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)


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Site- Directed Mutagenesis

For the constructs containing mutations at T-24, viable virusescould not be recovered.


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To confirm that failure of virus recovery was

not due to defective RNA synthesis:

CappedRNAtranscriptswere synthesized in vitro.

mRNA capping

Not-1 Linearized plasmid DNA

Capped RNA transcripts wereproduced

Arrow indicates full lengthRNA.

~5kb smaller transcript associated with transcription ofFCV

cloned genome

Lane 2- pQ14 parental plasmid

Gel analysisRNA stained

with EtBr

Lane 3-Y24A

Lane 4-Y24S

Lane 5-Y24T

Lane 6-Y24F


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To confirm that failure of virus recoverywas not due to defective protein

synthesis from mutagenized plasmids:

Capped RNA transcripts ->

Translated into RLL Labeled with methionine

Proteins were analyzed withSDS-PAGE.

Comparisons were madebetween wild type andmutations.

Lane 2 pQ14 parental plasmidLane 3 Y24ALane 4 Y24SLane 5 Y24T

Lane 6 Y24F

Lane 1 ispreviouslycharacterized

proteins fromTNT ofFCVORF1 clone

pTMF

-1 forcomparison


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To verify that failure to recover virus was

not due to aberrant transcription ortranslation

MVA/T7 infected CRFK cellswere transfected with wildtype and mutagenizedplasmids.

Proteins were radiolabeled.

Cell lysates were prepared andincubated with p-39 specific

serum precipitation of Ag-Abcomplexes with Sepharoseprotein A beads.

Analysis using SDS-PAGE andautoradioography

Mutants and wild-type transfections showedsimilar levels of mature p39 protein expressionusing MVA/T7.Thus, non-structural proteins were synthesized intransfected cells; however, structural proteinsynthesis did not occur.


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Comparison of growth properties of recoveredmutant viruses and wild-type viruses.

Growth kinetics of wild-type and mutant viruses were similar on plaqueassays 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 37C cells were washed and added with agaroseoverlay cells incubated at 37C for 24 hours in humidified CO2 incubator.

Monolayers fixed with formalin and stained with crystal violet.


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Growth characteristics were analyzed by measuring log virus titers.

After infection, the virustiter was determined bythe plaque assay.

Mutant viruses showedsimilar growth kinetics tothe wild-type virus.


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Examination of effects of VPg mutations on proteolytic processing ofthe ORF-1 polyprotein.

Cells infected with Y12A, Y76Aand Y104A or wild-type wereanalyzed by

immunoprecipitation withVPg- specific antibodies.

VPg precursors and 2 forms ofVPg were observed in thewild-type and three mutations(Y12A, Y76A and Y104A).

Thus, non-lethal mutations donot affect proteolyticprocessing of ORF1.


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Conclusion

Data in this study has shown that tyrosine 24 of

theF

CV VPg protein is essential forF

CVreplication.

mutagenesis of tyrosine 24 in the vpg protein

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