Department of Veterinary and Biomedical Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68503, USA
Correspondence
Clinton Jones
cjones{at}unlnotes.unl.edu
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ABSTRACT |
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Supplementary tables showing regulation of the IFN- promoter by IKK
and TBK1 and regulation of ISRE-dependent transcription by bICP0 are available in JGV Online.
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MAIN TEXT |
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The bICP0 protein activates viral gene expression and is encoded by IE transcription unit 1 (IEtu1) (Wirth et al., 1992; Everett, 2000
). bICP0 RNA is expressed constitutively during productive infection because the gene has an IE and E promoter (Fraefel et al., 1994
). bICP0 (Fig. 1
a) and herpes simplex virus type 1 (HSV-1) ICP0 proteins contain a well-conserved C3HC4 zinc RING finger, near their respective N termini, that is crucial for transcriptional activation (Everett, 1987
, 1988
; Everett et al., 1993
; Inman et al., 2001
). ICP0 (Maul et al., 1993
; Maul & Everett, 1994
; Everett et al., 1997
, 1999a
, b
) and bICP0 (Parkinson & Everett, 2000
; Inman et al., 2001
) colocalize with and disrupt the promyelocytic leukaemia protein-containing nuclear domains (ND10 or PODS).
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HSV-1 infection of cultured human cells induces interferon (IFN) production. ICP0, ICP34·5 and Us11 are the known viral genes that inhibit IFN activation after infection (Mossman et al., 2000, 2001
; Mossman & Smiley, 2002
; Peters et al., 2002
; Lin et al., 2004
). The viral glycoprotein gD induces IFN-
production in mononuclear cells, leading to IFN response factor 3 (IRF3) activation (Katze et al., 2002
). Mice lacking type I and type II IFN receptors in combination with RAG-2 gene deletions die within a few days of BHV-1 infection, whereas infection of wild-type (wt) mice does not lead to clinical symptoms (Abril et al., 2004
). To date, the BHV-1 genes that regulate IFN have not been identified.
To test whether bICP0 inhibited IFN-dependent transcription in the absence of other viral genes, transient-transfection assays were performed using a wt bICP0 construct, a zinc RING finger mutant (13G/51A) or a deletion mutant that lacks the last 320 aa of bICP0 (bICP0) (Fig. 1a
). These constructs express similar levels of bICP0 in transiently transfected cells (Zhang & Jones, 2001
; Henderson et al., 2004
; Zhang et al., 2005
). We predicted that bICP0 regulates IFN-dependent transcription because HSV-1 ICP0 inhibits IFN-dependent transcription (Mossman et al., 2000
, 2001
; Mossman & Smiley, 2002
; Lin et al., 2004
). Except for the zinc RING finger located near the N terminus of bICP0 (Fig. 1b
), there is little similarity between bICP0 and ICP0, making it necessary to test formally whether bICP0 inhibits IFN-dependent transcription.
The IFN- promoter was initially tested because it is activated strongly by early events that occur following virus infection (Katze et al., 2002
). For these studies, a plasmid containing the human IFN-
promoter (110 to +20) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was used. This promoter construct was obtained from Dr Stavros Lomvardas (Columbia University, NY, USA) and it contains elements that are activated by virus infection (Munshi et al., 2001
). As reported previously (Peng et al., 2005
), IFN-
promoter activity was low in mouse neuroblastoma cells (neuro-2A cells) (Fig. 1b
). Double-stranded RNA [polyinosinicpolycytidylic acid, poly(IC)] increased IFN-
promoter activity by more than 11-fold in neuro-2A cells (Fig. 1b
). bICP0 decreased IFN-
promoter activity in a dose-dependent manner and at the highest concentration of bICP0 tested, IFN-
promoter activity was reduced by approximately 10-fold. We also tested a plasmid that expresses the latency-related (LR) gene products because the LR gene overlaps bICP0 (Jones, 2003
). LR gene products were unable to inhibit IFN-
promoter activity in transiently transfected neuro-2A cells (Fig. 1b
).
IRF3 and IRF7 are transcription factors that are activated by phosphorylation following IFN induction (Barnes et al., 2002). Overexpression of IRF3 or IRF7 consistently stimulated IFN-
promoter activity by more than five- or sevenfold, respectively, in neuro-2A cells (Fig. 1c
). With as little as 1 µg bICP0 added to the cotransfection mix, IFN-
promoter activity was reduced by more than twofold when activated by IRF3 and by fivefold when activated by IRF7 (Fig. 1c
). When bICP0 was cotransfected with a BHV-1 LR promoter construct, bICP0 activated LR promoter activity by more than sevenfold, which confirmed the results of earlier studies (Bratanich & Jones, 1992
). These studies indicated that bICP0 inhibited IFN-
promoter activity, but activated LR promoter activity in transiently transfected neuro-2A cells.
IRF3 is activated by phosphorylation in two steps (Barnes et al., 2002; Sarkar et al., 2004
) and two protein kinases, IKK
and TBK1, coordinate IRF3 activation (Fitzgerald et al., 2003
; Sharma et al., 2003
). Cytomegalovirus expression plasmids containing these protein kinases were obtained from Dr Tom Maniatis (Harvard University, MA, USA). IKK
consistently activated IFN-
promoter activity in neuro-2A cells by more than sevenfold (Fig. 2
a and Supplementary Table S1a, available in JGV Online). wt bICP0 or the
bICP0 mutant reduced IFN-
promoter activity by approximately 50 % using 1 µg bICP0, and repression occurred in a dose-dependent fashion. At the higher concentration of bICP0 (wt or
bICP0), activation of IFN-
promoter activity by IKK
was negated. In contrast, the 13G/51A zinc RING finger mutant was unable to inhibit IFN-
promoter activity to basal levels. Similar results were obtained in human 293 cells (data not shown).
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Activated IRF3 induces expression of IFN-4 in mice or IFN-
1 in humans, and IRF3 cooperates with other transcription factors to activate the IFN-
promoter (Fitzgerald et al., 2003
; Sharma et al., 2003
; Sarkar et al., 2004
). Transcription of other IFN-
subtypes requires IRF7, which is crucial for type I IFN-dependent immune responses in mice (Honda et al., 2005
). IFN-stimulated response elements (ISREs) are present in many genes activated by IFN and are necessary for IFN induction. Consequently, we tested whether bICP0 inhibited ISRE-dependent transcription.
We subsequently tested whether bICP0 inhibited a minimal human immunodeficiency virus promoter construct with four consensus ISREs (pISRE). The ISRE elements in pISRE are from the ISG15 gene, and pISRE was obtained from Dr L. Zhang (University of Nebraska, NE, USA). IRF3 stimulated pISRE promoter activity by more than 50-fold in bovine cells (9.1.3), 40-fold in 293 cells or 15-fold in neuro-2A cells (Fig. 3a and Supplementary Table S2a). Activation of pISRE promoter activity by IRF3 was inhibited by almost threefold in 9.1.3 or neuro-2A cells and sixfold in 293 cells when 1 µg wt bICP0 was used in the transfection. The zinc RING finger mutant (13G/51A) was unable to effectively repress IRF3 induction of pISRE promoter activity in 9.1.3 cells. In 293 or neuro-2A cells, the 13G/51A mutant reduced pISRE promoter activity almost as efficiently as wt bICP0. The C-terminal bICP0 mutant (
bICP0) and wt bICP0 inhibited IRF3 induction of pISRE promoter activity with similar efficiency, suggesting that aa 1356 were sufficient for inhibiting pISRE promoter activity.
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Collectively, these studies indicated that bICP0 repressed the IFN- promoter (an early IFN response) and ISRE-dependent transcription (a relatively late IFN response). Conversely, bICP0 activated the LR promoter in 293, 9.1.3 and neuro-2A cells. These studies do not preclude the possibility that bICP0 inhibits other cellular promoters, nor do they explain how bICP0 inhibited IFN-
- or ISRE-dependent promoter activity. We suggest that, in general, interactions between bICP0 and cellular transcription factors stimulate viral transcription, but repress certain cellular promoters.
The two point mutations within the bICP0 zinc RING finger prevent activation of the thymidine kinase (TK) promoter and productive infection in all cell types that have been examined (Inman et al., 2001). In contrast, the zinc RING finger was only required to inhibit IRF3 induction of pISRE promoter activity in bovine cells (9.1.3) and IKK
induction of IFN-
promoter activity, suggesting that the zinc RING finger was necessary for inhibiting a specific IRF3-dependent step. HSV-1-encoded ICP0 also represses the antiviral effects of IFN (Mossman et al., 2000
; Mossman & Smiley, 2002
) by inhibiting IRF3 and IRF7 induction of IFN-stimulated genes, and the zinc RING finger is required for blocking activation (Lin et al., 2004
).
In all cell types examined, the bICP0 deletion mutant efficiently inhibited pISRE and IFN-
promoter activity, suggesting that aa 357676 were not important. The C terminus of HSV-1 ICP0 also does not play a major role in blocking IFN-dependent transcription (Lin et al., 2004
). The
bICP0 construct does not activate a simple HSV TK promoter (Inman et al., 2001
; Zhang & Jones, 2001
), suggesting that bICP0 sequences required for activating transcription are distinct from sequences that inhibit IFN-dependent transcription. A recent study has identified several functional domains within bICP0 that are necessary for activating a simple viral promoter (Zhang et al., 2005
). It will be of interest to identify the bICP0 functional domains that inhibit IFN-dependent transcription and to compare them with sequences necessary for activating viral transcription.
Although we believe that bICP0 is an important viral gene that inhibits the IFN response, it is likely that other BHV-1 genes antagonize the IFN response following infection. Support for this hypothesis comes from studies demonstrating that HSV-1 genes encoding ICP0, 34·5 and Us11 inhibit the IFN response (Mossman et al., 2000, 2001
; Katze et al., 2002
; Mossman & Smiley, 2002
). BHV-1 apparently does not encode a 34·5 homologue, suggesting that other BHV-1 genes with similar functions exist. Future studies will attempt to identify other viral genes that regulate the IFN response, and elucidate their role in pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 14 April 2005;
accepted 21 July 2005.
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