TheGeneEncoding theglll Envelope Protein ofPseudorabies VirusVaccine Strain Bartha Contains a Mutation Affecting Protein Localization

Pseudorabies virus (PRV)vaccine strain Bartha hasa diminished capacity tocausedisease andharbors a variety ofmutations affecting virulence. Ithasbeenreported thatPRVBarthaproduces virions withreduced amountsofthemajorenvelope glycoprotein gIH.Onehypothesis was thatthis phenotype was duetoreduced expression ofthegIII gene.Inthis report, we demonstrate thatthereduced amountofgIll invirions was not mediated atthelevel oftranscription, butrather reflected a defect inprotein localization. We describe experiments with genereplacement technology toprovethattheexpression defect was closely linked tothegIII geneitself. Usingpulse-chase experiments, we founda defect similar tothatobserved forcertain signal sequencemutations ofPRV BeckergIII. TheBarthagIIIprotein was translated, butwas inefficiently introduced intothemembraneprotein export pathway. Consequently, only a fraction oftheprimary Bartha gIlltranslation product was glycosylated andmatured. Theremaining fraction stayed presumably inthe cytoplasm, whereitneverbecameglycosylated orinserted intocell orvirus membranes. Theresult was that Bartha-infected cells produced virions withreduced amountsofgIII intheir envelopes. Comparison oftheDNA sequenceofthepromoter andamino-terminal coding regions ofBecker andBarthagIll genesrevealed asingle basepairdifference inBartha, changing codon14ofthesignal sequencefroma leucine (CTC)toa proline (CCC)codon. We suggest thatthesignal sequencemutation isresponsible fortheapparent reduced expression phenotype ofthis attenuated strain. Thismutation represents, toour knowledge, thefirst reported natural signal sequencemutation ina herpesvirus glycoprotein. Pseudorabies virus (PRV)isanalpha herpesvirus causing anatural infection inswine similar tothat ofherpes simplex virus inhumans(4,8).Because thedisease inswineisof economic importance, there hasbeenconsiderable effort to control this problem through vaccination programs. Accord- ingly, there hasbeenanincreased effort todevelop attenu- atedlive vaccine strains. Theseattenuated PRVstrains have beenstudied indetail byanumberoflaboratories tounder- stand thegeneral mechanisms ofherpesvirus virulence (1-3,