doi:10.1016/j.virol.2006.04.021 
Copyright © 2006 Elsevier Inc. All rights reserved.
Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression
aDepartment of Medical Microbiology and Immunology, University of Alberta, 632 Heritage Medical Research Center, Edmonton, Alberta, Canada T6G 2S2
Received 13 March 2006;
revised 31 March 2006;
accepted 14 April 2006.
Available online 5 June 2006.
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Abstract
HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.
Keywords: Herpes simplex virus; VP16; ICP0; Innate immunity; Somatic cell hybrid; Cell fusion; Reovirus p14
Fig. 1. Host range phenotype of KM110. HEL cells (stained red with CMTMR) and U2OS cells (stained blue with CMAC) were mixed and infected with 10 PFU/cell of wild-type HSV-1 KOS (A and B) or KM110 (C and D) for 9 h. Cells were then fixed and scored for ICP4 (A and C) or replicated viral genomes (B and D) via IF and DNA-FISH respectively (green signals). Representative fields of view are shown. Scale bar = 10 μm.
Fig. 2. Fusion does not affect the phenotype of U2OS or HEL cells. Homokarya formed between U2OS cells (A, stained red with methyl bromide) or HEL cells (B and C, stained green with CFMDA) were scored for their permissivity to KM110. Cells transfected with pcDNA3-p14 (A and B) or pcDNA3-p14 and the ICP0 expression vector pDR27 (C) were mixed 6 h posttransfection with a parallel culture that had been infected with KM110 (10 PFU/cell) 1 h previously. Anti-p14 antiserum was added after 2 h, and cells were fixed and scored for ICP0 via IF (blue) 7 h later. Representative homokarya are shown. Scale bar = 10 μm.
Fig. 3. Experimental design.
Fig. 4. The restrictive phenotype of HEL cells is dominant in heterokarya. HEL cells (red) were infected with 10 PFU/cell of KM110, then fused with p14-expressing U2OS cells (blue) 1 h later as outlined in Fig. 3. In panels B and D, the U2OS cells were transfected with the ICP0 expression vector pDR27 in addition to the p14 expression plasmid. Cells were fixed 9 h later and scored for ICP4 expression (A and B) and replicated viral DNA (C and D) by IF and DNA-FISH respectively (green signals). Representative heterokarya are shown. Scale bar = 10 μm.
Fig. 5. The KM110 genome responds to VP16 delivered in trans 1 h postinfection. HEL cells (red) were fused with U2OS cells (blue) 1 h after infection with KM110, as in Fig. 4. In panels B and D, the U2OS cells were transfected with the VP16 expression vector pKOS-VP16 in addition to the p14 expression plasmid. Cells were fixed 9 h later and scored for ICP4 expression (A and B) and replicated viral DNA (C and D) by IF and DNA-FISH respectively (green signals). Representative heterokarya are shown. Scale bar = 10 μm.
Fig. 6. Nuclear delivery of viral genomes is complete within 3 h. HEL cells (red) were mixed with U2OS cells (blue) expressing ICP0 and p14 at 1 (A and C) or 3 (B and D) h after infection with KM110, essentially as outlined in Fig. 3. Cells were fixed 9 h after fusion and scored for replicated viral DNA (green signal). The images shown in panels A and B were obtained in an experiment where the CMAC stain was lost from the U2OS nuclei; the images shown in panels C and D are from a separate experiment where nuclear CMAC staining was retained. Representative heterokarya are shown. Scale bar = 10 μm.
Fig. 7. Reactivation of the KM110 genome by ICP0 provided in trans 48 h postinfection. HEL cells (red) were infected with 10 PFU/cell of KM110, incubated 48 h, then fused with p14-expressing U2OS cells (blue) as outlined in Fig. 3. In panels B and D, the U2OS cells were cotransfected with the ICP0 expression vector pDR27. Cells were fixed 9 h later and scored for ICP4 expression (A and B) and replicated viral DNA (C and D) by IF and DNA-FISH respectively (green signals). Representative heterokarya are shown. Scale bar = 10 μm.
Fig. 8. VP16 is unable to reactivate KM110 at late times postinfection. HEL cells (red) were infected with 10 PFU/cell of KM110, incubated 48 h, then fused with p14-expressing U2OS cells (blue) as in Fig. 7 and as outlined in Fig. 3. In panels B and D, the U2OS cells were cotransfected with the VP16 expression plasmid pKOS-VP16. Cells were fixed 9 h later and scored for ICP4 expression (A and B) and replicated viral DNA (C and D) by IF and DNA-FISH respectively (green signals). Representative heterokarya are shown. Scale bar = 10 μm.
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