SV40 is thought to induce transformation in part by acting on key transcriptional regulators and thereby altering cellular gene expression. Thus, SV40 TAg antagonizes the ability of Rb proteins to repress E2F-dependent gene expression leading to the expression of genes required for cell cycle entry and progression while simultaneously blocking p53-dependent transcription and consequently inhibiting apoptosis. Consistent with this view, we previously reported that TAg upregulates E2F-dependent genes in both primary MEFs and transgenic murine enterocytes and that p53-dependent transcription was not induced in either of these systems (7). However, these studies were limited by the use of a Agilent mouse cDNA array which only included 8462 genes of the estimated 21,000 mouse genes.
We have now extended these studies by the use of mouse whole-genome arrays and by including three key mutants (TAgN136, TAg3213 and TAgD44N) in addition to TAgwt in the analysis. TAg3213 and TAgD44N are defective for Rb protein inactivation but retain the ability to bind p53, while TAgN136 is defective for p53 interaction but retains the ability to inhibit the Rb proteins. In addition, to eliminate genes whose regulation may be altered as a consequence of cell line establishment, we have analyzed gene expression in MEFs abortively infected with SV40.
TAg induces E2F-dependent transcription and blocks expression of p53- target genes
Studies of transgenic enterocytes expressing wild-type or mutant TAg demonstrated that nearly all TAg-dependent gene regulation in these cells can be explained by the inactivation of the Rb proteins and resultant upregulation of E2F-dependent gene transcription. Neither wild-type nor the mutant TAg's affected the expression of p53-regulated genes, consistent with the lack of p53 expression in this cell type (Markovics et al., 2005
In this report we examined the consequences of wild-type and mutant TAg's in MEFs. Consistent with our studies in enterocytes we found that TAg induction of E2F-dependent transcription depends on both a functional J domain and Rb-binding LXCXE motif, and that the first 136 amino acids of TAg, that contains both of these elements is capable of upregulating these genes. In contrast to enterocytes, MEFs upregulated p53-dependent genes in response to TAgN136. However, p53-dependent genes were not induced, or in some cases were repressed, by TAgwt and by TAg3213 and TAgD44N, each of which retains the ability to bind p53.
Induction of immune response genes by TAg requires the LXCXE motif and p53 binding
Previously we noticed that TAg regulated a set of immune response genes in MEFs and that these same genes were not regulated by TAg in transgenic enterocytes (7). The use of the whole genome mouse array in this study clearly shows the upregulation of a large number of immune response genes by SV40 T antigen (). These genes represent two major classes: (1) Interferon-stimulated genes (ISGs), such as the OAS family, MX1, ISG56, ISG54, ISG15, Cig5, GTPase, P200 gene family and PKR; and, (2) genes involved in interferon induction and signaling such as IRF-7, IRF-9, RIG-1, STAT1 and STAT2. This collection of genes was upregulated in both MEFs abortively infected with SV40 as well as in MEFs stably expressing TAg indicating that their altered regulation is not an artifact arising from cell line establishment.
The genetic data suggests that the immune response genes are regulated by a common mechanism. All of these genes are regulated by TAgwt and by TAgD44N but not by TAgN136or TAg3213. This suggests a requirement for both the LXCXE motif and a TAg function or functions carboxy-terminal to amino acid 136 The inability of TAg3213 to regulate the immune response genes suggests that TAg binding to Rb family members plays a role in this effect. However, the inactivation of Rb proteins by TAg is thought to be J domain dependent so the observation that TAgD44N is capable of regulating these genes argues against this possibility. Perhaps the LXCXE motif can also act on Rb proteins in a J-domain-independent manner. Alternatively, the LXCXE motif may target cellular proteins other than Rb and these unknown targets may play a role in regulating the immune response genes.
is fully able to inactivate Rb family members, yet it is unable to induce ISGs. This indicates that the regulation of immune response genes requires one or more activities residing in the carboxy-terminal region of TAg. One candidate activity is the ability of TAg to bind p53 and we found that a p53 binding defective mutant of TAg (Patch-1) is unable to upregulate ISGs (). This indicates that the TAg-p53 interaction is necessary for ISG induction. At present we can not distinguish between a “loss of function” model, in which p53 normally functions to repress ISG induction and TAg blocks this p53 action, or a “gain-of-function” model in which the TAg-p53 complex is actively involved in ISG expression. Bocchetta et al
have shown that TAg-p53 complexes are required to activate the insulin-like growth factor-I promoter (Bocchetta et al., 2008
). Consistent with a “gain of function” hypothesis, enterocytes, which lack p53 expression, do not show induction of immune response genes (Cantalupo et al., 2009
; Rathi et al., 2009
). In either case, binding of TAg to p53 alone is not sufficient for ISG induction as TAg3213
can bind to p53 but is unable to induce the immune response genes. Collectively, our mutant analysis suggests cooperation between LXCXE motif of TAg and binding with p53 in the regulation of immune response genes.
TAg activates STAT-1 in the absence of interferon production
Interestingly, TAg is capable of inducing the downstream interferon pathway without affecting the levels of IFN-α or IFN-β. We found that TAg induces STAT1 Tyr701 phosphorylation suggesting that TAg can activate the interferon signaling pathway independent of interferon production. One possibility is p53-dependent activation of STAT1 by c-Abl1 tyrosine kinase instead of the classical JAK-STAT pathway as reported by Youlyouz-Marfak et al (Youlyouz-Marfak et al., 2008)
. Furthermore, TAg mutants capable of inducing ISG expression also induce STAT-1 phosphorylation while mutants defective for ISG induction do not induce STAT-1 phosphorylation. This suggests that the primary mechanism by which TAg induces ISG's is through STAT-1 signaling.
In conclusion, we have shown that SV40-transformed MEFs have activated the interferon pathway in the absence of interferon production. The biological consequences of this activation remain unclear. However, one practical consequence of this observation is in the common use of TAg in immortalization of MEFs obtained from knockout mice. Our observation clearly demonstrates that this practice should be critically evaluated while testing functional consequences of gene knockouts, especially in the studies involving genes that modulate innate immune signaling pathways.
Several herpesviruses, such as Epstein–Barr virus, herpes simplex virus, Kaposi's sarcoma-associated herpesvirus and HCMV have been shown to activate IFN-responsive genes, such as MxA and OAS (Browne et al., 2001
; Mossman et al., 2001
; Poole et al., 2002
; Ruvolo et al., 2003
; Zhu et al., 1998
). However, in these cases the induction occurs in the context of a productive infection and the effects of the ISG's are later mitigated by other viral functions. One interesting question that arises is how TAg expressing MEFs are able to survive in the presence of high levels of ISGs which are known to create growth inhibitory environment. Future studies are needed to identify the cellular target(s) on which TAg acts to elicit ISG expression and to explore the connection between ISGs and transformation by TAg.