Downloaded from www.microbiologyresearch.org by IP: 23.22.50.124 On: Mon, 18 Apr 2016 04:15:48 Journal of General Virology (1998), 79, 1603–1611. Printed in Great Britain ................................................................................................................................................................................................................................................................................... The UL4 gene of herpes simplex virus type 1 is dispensable for latency, reactivation and pathogenesis in mice Patricia Y. Jun, 1 Lisa I. Strelow, 1 † Ronald C. Herman, 2 Howard S. Marsden, 3 Truls Eide, 4 Lars Haarr 4 and David A. Leib 1,5 1,5 Department of Ophthalmology and Visual Sciences 1 and Department of Molecular Microbiology 5 , Washington University School of Medicine, 660 South Euclid Avenue, Box 8096, St Louis, MO 63110, USA 2 Calydon Inc., Menlo Park, CA 94025, USA 3 MRC Virology Unit, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK 4 Centre for Research in Virology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway The UL4 gene of herpes simplex virus type 1 is predicted to encode a 215 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed Introduction In humans and in experimental animals the pathogenesis of herpes simplex virus type 1 (HSV-1) occurs in a series of discrete stages (Wildy et al., 1982). Acute virus replication at the periphery is followed by virus entry into neuronal termini. Intra-axonal transport moves the virus to sensory ganglia, where further replication may occur and latency is established. The viral genome is episomal within neuronal nuclei, and the only abundant viral gene products expressed during latency are the latency-associated transcripts (Stevens et al., 1987). Latency may periodically break down in response to certain Author for correspondence : David Leib (at Department of Ophthalmology and Visual Sciences). Fax 1 314 362 3638. e-mail Leibam.seer.wustl.edu † Present address : Department of Molecular Microbiology & Immunology, Oregon Health Sciences University, Portland, OR 97201-3098, USA. a 23 kDa band in extracts of wild-type and marker- rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C 3 H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice. stimuli, leading to virus reactivation and shedding at the periphery. The molecular mechanisms responsible for the altered regulation of viral gene expression during latency remain poorly understood. A number of studies, however, have indicated that increased intracellular cAMP levels during latency may play a direct role in triggering the expression of a variety of cAMP-responsive viral genes, thereby leading to reactivation (Bloom et al., 1997; Deb et al., 1993; Leib et al., 1991 ; Wheatley et al., 1992). A perfect consensus cAMP- responsive element (CRE) exists 124 bp upstream of the UL4 gene, 64 bp upstream from its putative TATA box, although it is not known if this CRE is functional. HSV-1 encodes at least 74 proteins, but the function of many of them remains unknown (McGeoch & Schaffer, 1993). Targeted mutagenesis of HSV-1 has demonstrated that many such genes are dispensable for virus replication in cell culture (Baines & Roizman, 1991). Experiments in which viruses with mutations in dispensable genes are used in animal models of neurovirulence, latency and reactivation have revealed that 0001-5413  1998 SGM BGAD