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 (neoR) 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-neoR 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.
Department of Pathology, University of Cambridge, UK.
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Lachmann
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Brown
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Efstathiou
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D019868:Kanamycin Kinase; D051379:Mice; D008807:Mice, Inbred BALB C; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics*; D014158:Transcription, Genetic; D014709:Vero Cells; D017735:Virus Latency
D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D012318:RNA Polymerase I