Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, UK1
Author for correspondence: Chris Preston. Fax +44 141 337 2236. e-mail c.preston{at}vir.gla.ac.uk
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Cellular interferon-responsive gene expression is induced upon infection of human fibroblasts with human cytomegalovirus (HCMV). The effect was observed even in the absence of de novo protein synthesis, indicating that it is mediated by virion components (Zhu et al., 1997 , 1998
; Navarro et al., 1998
), and binding of glycoprotein B to the cell surface is sufficient to elicit the response (Boyle et al., 1999
). There are differences in the details of the reports describing induction by infection with HCMV: in one case a number of interferon-responsive genes, including those encoding cellular genes named ISG54, ISG15 and 9-27, were activated (Zhu et al., 1997
, 1998
), whereas in the other ISG54 was switched on but ISG15 and 9-27 were not (Navarro et al., 1998
). Activation of ISG54 synthesis correlated with the formation of a novel protein complex that bound to sequences in the promoter of this gene, suggesting that, as in the case of paramyxovirus infection, induction by HCMV and by interferon itself does not follow exactly the same signalling pathway (Navarro et al., 1998
). In the report of Zhu et al. (1997)
, infection with herpes simplex virus type 1 (HSV-1) gave only very small increases in the level of ISG-specific RNAs, leading to the conclusion that HSV-1 does not significantly affect the expression of this set of cellular genes. We have re-examined the expression of interferon-responsive genes in cells infected with HSV-1, by using mutants impaired for immediate early (IE) transcription and by carrying out infection in the presence of cycloheximide to block viral protein synthesis.
Human foetal lung (HFL) fibroblasts, the human fibrosarcoma line 2fTGH, and mutants of 2fTGH named U1A (mutant for Tyk2), U3A (mutant for STAT1) and U4A (mutant for JAK1) were used (Pellegrini et al., 1989 ; McKendry et al., 1991
; Darnell et al., 1994
). HCMV was strain AD169 and wild-type HSV-1 was strain 17. The HSV-1 mutants in1814, specifying a defective virion transactivator VP16, and in1312, which has mutations that inactivate VP16 and the IE transactivator proteins ICP0 and ICP4, have been described previously (Ace et al., 1989
; Preston et al., 1998
). Mutant in1312 is severely impaired for viral IE gene expression, and cells can survive infection with this virus at relatively high m.o.i. (up to an effective dose of 5 p.f.u. per cell: Preston et al., 1997
; Homer et al., 1999
). Probes specific for ISG54, ISG15 and 9-27 were prepared by RTPCR amplification of RNA isolated from HFL cells that had been treated for 6 h with human lymphoblastoid interferon-
(Sigma). The probe for IFI56 was a 270 bp BglIISspI fragment from a cloned copy of the gene (Chebath et al., 1983
). A probe for MxA was cleaved from a plasmid containing the cloned cDNA, kindly supplied by F. Weber (Institute of Virology, Glasgow, UK). A probe specific for glyceraldehyde phosphate dehydrogenase (GAPDH) was purchased from Ambion.
Monolayers of 2x106 to 1x107 HFL cells were infected with in1312 (effectively 5 p.f.u. per cell, although the measured titre was lower than this value due to the ICP0 mutation; the number of particles added was equivalent to that when 5 p.f.u. of wild-type HSV-1 was used), wild-type HSV-1 (5 p.f.u. per cell) or HCMV (1 p.f.u. per cell), with 50 µg cycloheximide/ml in some cases, and incubated at 38·5 °C for 6 h. Total cytoplasmic or polyadenylated RNA was isolated, separated on formaldehydeagarose gels, blotted and hybridized to radiolabelled gene-specific probes (Nicholl & Preston, 1996 ) (Fig. 1
). Monolayers were mock infected with cycloheximide added (lane 1), treated with 103 units human lymphoblastoid interferon-
/ml, plus cycloheximide (lane 2), or infected with in1312 plus cycloheximide (lane 3), in1312 without cycloheximide (lane 4), wild-type HSV-1 with cycloheximide (lane 5) or HCMV with cycloheximide (lane 6). Hybridization with probes specific for ISG54 and GAPDH, a control gene that was not expected to be affected by interferon-
treatment, demonstrated that strong induction of ISG54-specific RNA, comparable to that after addition of interferon-
, was observed after infection with all viruses tested in the presence of cycloheximide, and with in1312 in the absence of the inhibitor. In a second set of experiments, HFL cell monolayers were infected with wild-type HSV-1 with or without cycloheximide. Infection in the absence of cycloheximide did not result in increased ISG54 mRNA levels, in agreement with the findings of Zhu et al. (1997)
(Fig. 1
, lane 9), whereas strong induction was observed when the inhibitor was present (Fig. 1
, lane 8). Induction of ISG54 occurred after infection with in1814 that had been gradient purified by the method of Szilagyi & Cunningham (1991)
(Fig. 1
, lane 12), suggesting that components of the virus rather than contaminating cellular material were responsible, and the effect was not diminished by heavy UV-irradiation of the in1814 preparation to reduce its titre by a factor of 5x105 (Notarianni & Preston, 1982
), confirming that viral protein synthesis is not required for induction (Fig. 1
, lane 11). To determine whether infection stimulated the release of soluble factors (for example, interferon itself), the medium from cells infected with in1312 at 38·5 °C for 6 h was centrifuged at 25000 g for 1 h to remove virions and applied to fresh HFL monolayers. This procedure did not result in the induction of ISG54 (results not shown).
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The observation that ISG54-specific transcripts were not detected after normal infection with HSV-1, as opposed to the situation when cycloheximide was present from the time of infection, suggests that, as is the case for other viruses, HSV-1 encodes functions that inhibit the interferon response. It is likely that the relevant virus-specified gene products have a role in combating the interferon-mediated host defence to HSV-1, which has an important bearing on virus pathogenesis (Leib et al., 1999 ).
Paramyxovirus infection activates transcription from the promoter of ISG15, but not that of 9-27, through a novel signalling pathway involving phosphorylation and recruitment of the cell factors IRF-3 and IRF-7 (Wathelet et al., 1998 ; Guo et al., 2000
). The mechanism used by paramyxoviruses is unlikely to account entirely for the effects of HSV-1 or HCMV, since these herpesviruses do induce 9-27, albeit at least 10-fold relatively less strongly than they induce ISG15. The results may therefore highlight further variation in the ways in which cells respond to infection by different viruses.
The experiments described here report a novel feature of infection with HSV-1. It has been shown previously that HSV-1 induces the transcription of certain cellular genes, in some cases as a very early event in infection (Notarianni & Preston, 1982 ; Kemp et al., 1986 a
, b
; Preston, 1990
; Hobbs & DeLuca, 1999
). The most relevant report demonstrated the presence of a new protein of Mr approximately 56000 after treatment of human cells with an HSV-1 mutant blocked at an early stage of infection (Preston, 1990
), and it is tempting to speculate that this represented one or more members of the ISG54 family.
Our results additionally show that noncytotoxic prototype HSV-1 vectors, or amplicons, that are severely impaired for viral gene expression may nonetheless alter cellular gene expression. It is not yet clear whether these changes affect cell physiology in any significant way, or if they have an influence on the cellular response to infection by HSV-1 or other viruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 March 2000;
accepted 19 May 2000.