Axxima Pharmaceuticals AG, Max-Lebsche-Platz 32, 81377 Munich, Germany
Correspondence
Matt Cotten
matt.cotten{at}axxima.com
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ABSTRACT |
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Supplementary material available in JGV Online.
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INTRODUCTION |
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The HCMV immediate-early (IE) gene promoter encodes several functional NF-B binding sites and the promoter function is increased by NF-
B activation (Sambucetti et al., 1989
; Prosch et al., 1995
). Furthermore, HCMV encodes and expresses proteins that can themselves activate the NF-
B pathway (Yurochko et al., 1995
, 1997a
, b
). For these reasons, one could conclude that NF-
B activation plays a positive role for HCMV replication and that inhibiting NF-
B activation would be a useful strategy for blocking virus replication. Under some conditions and in some cell types, NF-
B activation is important for anti-apoptotic, cellular repair and survival pathways, and perhaps the viral activation of NF-
B is a viral mechanism to avoid cellular apoptosis that would limit virus replication and spread. However, NF-
B is also an important transcription factor for a number of cellular anti-viral genes including inflammatory and anti-viral cytokines and genes whose products are part of the host innate immune response. Because of the evolutionary potential of viruses, it is quite possible that NF-
B is primarily a transcription factor activated in response to pathogen entry and important for driving anti-pathogen responses, but viruses have evolved to take advantage of the strongly and rapidly induced transcription factors available during infection. The virus must then evolve functions to block the inhibitory consequences of this NF-
B activation.
Recently the requirement for NF-B in cytomegalovirus replication has been examined using specific methods that assess the role of the transcription factor in the entire replication process (Benedict et al., 2004
). These investigators provided evidence that viral transcription and replication proceed normally when either the NF-
B pathway is blocked or when established NF-
B binding sites in the viral genome are eliminated.
We are interested in defining the host signal transduction pathways used by HCMV for replication. This is an important study, both for understanding how HCMV replicates as well as for identifying new targets for pharmaceutical intervention of this replication. Accordingly, we have examined the role of NF-B activation during the entire replication cycle of HCMV to distinguish if this activation is a pro-viral event (which would be consistent with a number of transcriptional studies of HCMV which, however, fail to examine the full HCMV life cycle) or if NF-
B activation drives an anti-viral response in the HCMV-infected cell.
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METHODS |
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Recombinant adenovirus vector construction and purification.
The recombinant adenoviruses used in this study were E1, E3 defective derivatives of adenovirus type 5 (reviewed by Russell, 2000). The cDNAs of desired proteins were cloned into a transfer plasmid; expression was driven by a HCMV IE promoter/enhancer and mRNA stability and polyadenylation were directed by a rabbit
-globin intron/polyadenylation signal. This expression cassette was introduced into a bacterial plasmid-borne adenovirus genome using recombination in bacteria (Chartier et al., 1996
; Michou et al., 1999
). Virus replication was established by transfecting the genome into the E1 complementing 293 cells (Graham et al., 1977
). The IKK
cDNA was isolated by PCR from a human kidney cDNA preparation and the sequence is identical to GenBank AF080157 and BD062400. In the plasmid expression construct and in the adenovirus the open reading frame contains a carboxy-terminal strep-tag. The kinase inactive IKKki mutant was generated by mutating the codon for lysine residue 44 (AAG) to an alanine codon (GCG). The I
B
Ser 32, 36 Ala mutation (termed I
B
here) is the same as described previously (Brown et al., 1995
; Traenckner et al., 1995
). Adenoviruses were purified from cell lysates using CsCl density-gradient centrifugation as described previously (Cotten et al., 1996
); virus was quantified using protein content (Cotten et al., 1996
) with the conversion factor 1 mg ml1 pure virion protein=3·4x10e12 viral particles ml1 (Lemay et al., 1980
). Control adenovirus was AdJ5, an Ad5 vector with the same viral genotype (E1, E3 defective) but lacking an expression cassette.
Reporter assays.
NF-B activation was monitored using a minimal promoter bearing multiple NF-
B binding sites driving a luciferase cDNA as previously described (Chiocca et al., 1997
). Briefly, 5x104 U373 (or HFF) cells were seeded in 96-well plates and were transfected with 275 (170) ng p3K NF-
B luciferase reporter plasmid and 75 (30) ng pGFP for normalization plus pI
B
or control plasmid. One day later, the cells were treated with the indicated reagents. Luciferase activity was then detected using standardized extracts with the LucLite plus luciferase detection system (Packard BioSciences) and normalized with GFP expression. Alternatively, a stable NIH3T3 cell line bearing the same luciferase construct was used (see Fig. 1
).
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Interferon- (IFN) induction.
IFN- induction was measured using real-time PCR with an ABI PRISM 7000 sequence detection system and the following primers: IFN-
, 5'-GACATCCCTGAGGAGATTAAGCA-3' and 5'-GGAGCATCTCATAGATGGTCAATG-3'; hybridization probe: 5'-FAM-CGTCCTCCTTCTGGAACTGCTGCAG-TAMRA-3'. GAPDH was quantified using an established TaqMan assay kit (ABI Prism TaqMan assay reagent for human GAPDH; Applied Biosystems).
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RESULTS |
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To increase the reliability of our conclusions, an alternative method of modulating NF-B activation was sought. Glucocorticoids, such as Dex, are potent inhibitors of NF-
B signalling with at least three mechanisms functional in this activity (reviewed by McKay & Cidlowski 1999
; Almawi & Melemedjian, 2002
). The activated glucocorticoid receptor can promote transcription of the inhibitor of NF-
B (I
B), can form complexes with p65, recruiting transcriptional inhibitory histone deacetylase to the promoters of NF-
B target genes, and can sequester limiting components of the transcriptional machinery to impair NF-
B activated gene expression.
To demonstrate that Dex could block NF-B signalling, cells were exposed to Dex and infected with HCMV or treated with NF-
B activating levels of TNF-
or IFN-
. Similar to the results obtained with the expression of I
B
, Dex treatment of U373, NIH3T3 or HFF cells clearly blocks the activation of NF-
B (Fig. 1b and 1c
, right panel). Thus, two distinct methods of blocking the NF-
B activation are available: the expression of a I
B
directed by recombinant adenovirus and the pharmacological inhibition of NF-
B signalling by Dex.
That HCMV infection itself activates NF-B has been known for some time (Kowalik et al., 1993
). Several distinct mechanisms have been described for this activation, including upregulation of both p105/p50 and p65 and relB expression at the transcriptional level by HCMV early gene expression (Yurochko et al., 1995
, 1997b
; Jiang et al., 2002
). The activation of NF-
B by HCMV can occur independently of virus replication and the inhibition of this function by pertussis toxin suggests a role for G-protein activation (Carlquist et al., 1999
). The activation of NF-
B by HCMV infection is demonstrated in Fig. 1(a) and (b)
, four sample sets, and Fig. 1(c)
. Importantly, both I
B
expression (Fig. 1a and c
) and Dex treatment (Fig. 1b and c
) block the HCMV-stimulated NF-
B activation. Interestingly, UV inactivation of the virus (which should eliminate viral gene expression) leads to slight increase in the NF-
B activation (see supplementary material Fig. S1), consistent with the published report that HCMV encodes gene products which counteract NF-
B signalling and block the induction of an innate immune response (Browne & Shenk, 2003
).
The replication of HCMV in cell culture can be inhibited by treating cells with either TNF- (Ito & O'Malley, 1987
; Manor & Sarov, 1988
; Paya et al., 1988
) or IFN-
(Yamamoto et al., 1987
; Torigoe et al., 1993
; Bodaghi et al., 1999
). Both cytokines activate NF-
B (see Fig. 1
); however, in the case of HCMV replication, the exact role of NF-
B in the cellular anti-viral responses activated by TNF-
and IFN-
has not been examined directly. We first demonstrated that under the conditions used here, treatment with either agent inhibited HCMV replication (Fig. 2
). TNF-
, and to a lesser extant IFN-
, inhibited HCMV replication comparably with high level ganciclovir (GCV) treatment (Fig. 2b
), which inhibits the virus by targeting a viral kinase and DNA polymerase. To investigate the requirement for NF-
B activation by the cytokines in the HCMV inhibition, I
B
or Dex were tested for their ability to modulate the HCMV inhibitory pathway activated by TNF-
and IFN-
. Both I
B
and Dex reduced the anti-viral effects of TNF-
and IFN-
on HCMV replication (Fig. 2
). Control transduction with adenovirus AdJ5 bearing an empty expression cassette does not influence the HCMV replication assay (see Eickhoff et al., 2004
and Fig. 2c, d
). However, as expected, Dex (Fig. 2b
) did not reverse the GCV inhibition of HCMV, which is not predicted to require NF-
B for function. Thus, two different anti-viral cytokines, TNF-
and IFN-
, both activate NF-
B and inhibit HCMV replication. Inhibition of NF-
B activation by either expression of I
B
or treatment of cells with Dex prevented the inhibitory activity of the two cytokines. Very similar results were obtained when virus replication was assayed by traditional plaque titration (Fig. 2c, 2d
) confirming the validity of the GFP assay results. (For a direct comparison between the plaque titration- and the GFP-replication assay, see supplementary Fig. S2.) Thus, the activation and function of the transcription factor NF-
B is essential for the inhibition of HCMV replication by TNF-
and IFN-
.
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The activation pathway of NF-B is primarily driven by stimuli that activate the IKK complex comprising the kinases IKK
and IKK
and the non-catalytic subunit IKK
/NEMO (reviewed by Ghosh & Karin, 2002
). Overexpression of IKK
with no additional stimulus was shown to be sufficient to activate the NF-
B pathway (Woronicz et al., 1997
). This results in phosphorylation of both I
B as well as p105 with ensuing processing, and p65 or p50 release and NF-
B activation (Lang et al., 2003
). Consistent with the literature, overexpression of IKK
alone activates NF-
B dependent gene expression and will inhibit HCMV replication (Eickhoff et al., 2004
), although in this published report, the control demonstrating that kinase activity was indeed required, was lacking. We examined this phenomenon in more detail here, including the appropriate control: a similar adenovirus expressing a kinase inactive point mutant of the kinase (Fig. 4
). Expression of wild-type IKK
provides a dose-dependent increase in NF-
B dependent gene expression (Fig. 4a
, lanes 25) while expression of a kinase inactive version of IKK
no longer activates NF-
B (Fig. 4a
, lanes 69). When tested on HCMV replication, adenovirus-directed expression of IKK
results in the expected inhibition of replication (Fig. 4b
, lanes 35), similar to the inhibition observed with the more pleiotrophic NF-
B activators TNF-
and IFN-
(Fig. 2
). We tested the IKK
K44R mutant for its capacity to inhibit NF-
B signalling, as this mutant has been proposed to inhibit HCMV-induced NF-
B activation in fibroblast cells (Caposio et al., 2004
), yet its effect on a full HCMV replication cycle has not yet been examined. Importantly, expression of comparable levels of kinase inactive IKK
K44R mutant does not impair HCMV replication (Fig. 4b
, lanes 68), consistent with the demonstration that the mutant does not activate NF-
B (Fig. 4a
). Transduction with an empty adenovirus of the same genotype, but lacking an expression cassette (AdJ5) is also not inhibitory to HCMV replication under these conditions (Fig. 4b
, lane 2).
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Either control fibroblasts or fibroblasts in which the NF-B pathway was blocked using I
B
, were infected with HCMV. The supernatants from these cells were then collected and tested for their ability to inhibit HCMV replication in fresh monolayers (Fig. 5
). The supernatant from HCMV-infected cells was inhibitory to HCMV replication in fresh cells (Fig. 5
, lanes 1, 3), and this inhibition was not significantly altered by transduction of the original cells with a control adenovirus (AdJ5, Fig. 5
, lanes 3, 4). However, if the original cells are transduced with increasing amounts of AdI
B
(and thus have blocked NF-
B signalling) the resulting supernatants showed decreasing capacity to inhibit HCMV with increasing I
B
(Fig. 5
, lanes 57). These results are consistent with the previous demonstration that the function of HCMV inhibitory cytokines is blocked when NF-
B signalling is blocked (Fig. 2a and b
).
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DISCUSSION |
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There are a series of relevant observations of the ability of glucocorticoids, potent NF-B inhibitory agents, to promote HCMV or murine CMV replication in cell culture, as well as in patients and in mice (Jordan et al., 1977
; Velasco et al., 1984
; Tanaka et al., 1984
; Koment, 1985
, 1989
; Forbes et al., 1990; Lathey & Spector, 1991
). We demonstrate here that two different methods of blocking NF-
B activation reverse the inhibitory effects of IFN-
and TNF-
. These results provide a molecular mechanism to explain a number of older literature reports on the stimulatory effects of glucocorticoids on HCMV replication and pathology.
Although these results are clearly surprising in light of earlier work examining HCMV IE promoter function and NF-B activation, they are consistent with more recent publications on this phenomenon. Benedict et al. (2001)
had previously found that HCMV replication was not impaired in cells expressing a non-degradable I
B mutant; this was the first demonstration that inhibition of NF-
B did not produce a barrier to HCMV. Furthermore, Eickhoff et al. (2004)
showed that the kinase RICK induces IFN-
via NF-
B signalling, which leads to inhibition of HCMV growth. In addition, Benedict et al. (2004)
demonstrated that the NF-
B sites in the IE promoter are not essential for either murine CMV or HCMV replication, and that both viruses grow in the presence of a non-degradable I
B or in p65/ fibroblasts. Using an alternative approach, we show in the current work that three different methods of manipulating NF-
B support the same conclusions: functional NF-
B activation is not only dispensable for productive HCMV replication in fibroblasts, but rather an essential part of an anti-viral host defence mechanism, which is mediated by IFN-
production. Blocking NF-
B by glucocorticoid or I
B does not impair replication, but blocks the inhibitory effect of IFN-
or TNF, which has in vivo relevance for the control of virus growth, on HCMV replication and hampers host cell anti-viral response.
The response of a particular gene to NF-B activation is influenced by the collection of other transcription factors that can be recruited to the promoter, the tissue expression of these transcription factors, local chromatin structure, as well as the strength, duration and nature of the NF-
B stimulus. It is clear that there are different kinetics of induction, strengths of expression, tissue expressions governed by different forms of NF-
B subunits expressed in the target cells as clearly demonstrated by Hoffmann et al. (2003)
. Furthermore, the NF-
B-dependent induction of the three isoforms of I
B can also strongly influence the resulting gene expression (Hoffmann et al., 2002
). Thus, it is an oversimplification to consider all NF-
B activation events as equal. It is possible that some low-level NF-
B activation, promoting a cell survival, might be beneficial to HCMV replication, and these subtleties would not appear in our studies. Alternatively, it is possible that HCMV replication in other cell types might have a different response to NF-
B activation. Certainly the survival role of the NF-
B pathway can have profoundly different importance in, for example, lymphocytes compared with fibroblasts. Thus, the NF-
B activation driven by HCMV capsid protein binding (Boyle et al., 1999
; Compton et al., 2003
) or by US28 (Casarosa et al., 2001
; Waldhoer et al., 2002
) may serve an important role in the survival of infected lymphocytes. In the evolution of the virus, this cell survival must be balanced against whatever negative consequences NF-
B activation may generate. It is now becoming clear that viruses encode mechanisms to block the inhibitory consequences of NF-
B activation, such as the ability of HCMV pp65 to impair NF-
B and IRF1 signalling (Browne & Shenk, 2003
). These strategies may allow the virus to take advantage of the positive features of activating NF-
B while preventing the negative consequences.
Finally, it should be noted that in vivo HCMV infection is not limited to fibroblasts. A full understanding of NF-B in HCMV function in vivo requires an analysis in other, clinically relevant, cell types. There is evidence that HCMV can reside latently in monocytic cell lineages (Soderberg-Naucler et al., 1997
; Pollock et al., 1997
), and it is possible that NF-
B activation may contribute to reactivation of virus replication. Indeed, Soderberg-Naucler and colleagues (1997)
have shown that IFN-
and TNF-
can promote HCMV reactivation in monocytes and this facet of the HCMV replication cycle must be considered. However, for the development of anti-HCMV strategies, it is becoming clear that blocking NF-
B function is not an effective method of impairing the replication of this virus.
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ACKNOWLEDGEMENTS |
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Received 20 July 2004;
accepted 28 October 2004.
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