The ICP22/ICP27 hybrid gene in equine herpesvirus 1 defective interfering particle-enriched infection

The genomes of equine herpesvirus 1 (EHV-1) defective interfering (DI) particles that mediate persistent infection were shown to encode a unique hybrid open reading frame composed of sequences that encode the 196 N-terminal amino acids of ICP22 linked in-frame to the C-terminal 70 amino acids of ICP27. Since this hybrid protein (designated as ICP22/ICP27) contains portions of two EHV-1 early regulatory proteins, its expression and function in EHV-1 gene regulation and DI particle-mediated persistent infection were investigated. Reverse transcriptase-PCR and S1 nuclease protection assays demonstrated that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western-blot analyses with antisera to the ICP22 and ICP27 proteins revealed that the 31-kDa ICP22/ICP27 hybrid protein was synthesized in the DI particle-enriched infection, but not in standard virus infection. Upon EHV-1 superinfection, the ICP22/ICP27 hybrid protein expressed from plasmid vectors significantly reduced expression of a reporter gene under the control of the EHV-1 immediate-early (IE) gene promoter and early gene promoters such as ICP27 and thymidine kinase. In uninfected cells, the ICP22/ICP27 hybrid protein moderately down-regulated the IE and ICP22 promoters, up-regulated late gene promoters such as IR5, and altered the regulatory function of the IE and ICP22 proteins in cotransfected cells. Recombinant EHV-1 DI particles that harbor and express the lacZ marker gene were generated. Infection of RK cell cultures with EHV-1 enriched with recombinant DI particles of known genotype generated colonies of persistently infected cells. The ability to generate persistently infected colonies varied significantly among recombinant DI particles containing different genomic structures. However, one recombinant DI particle strain that expressed the ICP22/ICP27 hybrid protein generated few persistently infected colonies, while disruption of the hybrid gene on another DI particle genome only moderately reduced its ability to produce persistently infected colonies under high multiplicity conditions. Overall, this study demonstrated that EHV-1 DI particles encoded and expressed a unique ICP22/ICP27 hybrid gene, which could significantly alter EHV-1 gene expression in DI particle-enriched infection but might not be essential for the establishment of DI particle-mediated persistent infection.