A murine RNA polymerase I promoter inserted into the herpes simplex virus type 1 genome is functional during lytic, but not latent, infection

The development of herpes simplex virus as a vector for neuronal gene delivery is dependent upon the identification and characterization of promoter elements capable of driving long-term expression during latency, The majority of RNA polymerase II (pol II) promoters studied are active during acute infection but silenced during latency, In order to investigate the potential of a murine RNA polymerase I (pol I) promoter to drive reporter gene expression during lytic and latent infection, we describe the construction and characterization of two recombinant viruses; SC16 LAT neo and SC16 US5 neo. These viruses contain a pol I-encephalomyocarditis virus internal ribosome entry site (EMCV IRES)-neomycin phosphotransferase gene (neo(R)) cassette inserted into the non-essential major latency associated transcript (LAT) and US5 regions respectively, Pol I promoter activity could be detected in the rodent BHK cell line, but not the primate derived Vero cell line - consistent with the known species specificity of such promoters, This activity was specific to a virus containing an active pol I promoter, However, in situ hybridization analyses of latently infected cervical dorsal root ganglia failed to detect pol I mediated transcription of the reporter gene indicating that the murine pol I promoter is silenced following the establishment of latency. Insertion of the pol I-EMCV IRES-neo(R) cassette into the major LAT locus resulted in the production of a hybrid LAT transcript during latency which was translocated to the cytoplasm of latently infected neurones.