The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus
© Haugh et al.; licensee BioMed Central Ltd. 2014
Received: 3 February 2014
Accepted: 23 May 2014
Published: 28 May 2014
The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/β) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response.
Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx.
These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV.
Avian Sarcoma Virus (ASV) is a prototypic alpharetrovirus (family Retroviridae) that can be pseudotyped to transduce mammalian cells and study host antiviral responses. We have previously shown that the cellular scaffolding protein Daxx, originally identified a mediator of death-receptor-triggered apoptosis , is also a potent anti-ASV restriction factor . Daxx is recruited to viral DNA by ASV integrase, where it promotes the rapid epigenetic repression of integrated viral DNA via recruitment of gene-repressive histone deacetylases (HDACs) and DNA methyl transferases [2, 3]. We identified an essential role for Daxx in controlling ASV replication by demonstrating that genetic ablation or RNA interference-mediated knockdown of Daxx expression resulted in significantly-increased expression of an ASV-encoded reporter gene [2, 3].
Type I (predominantly α/β) interferons (IFNs), are a family of cytokines with powerful antiviral and immune-modulatory effects, and are rapidly induced in most cells upon virus infection. Once produced, IFNs activate an antiviral state in the infected cell, as well as in surrounding cells, by Jak/STAT-regulated induction of >1000 IFN-stimulated genes (ISGs) [4, 5]. Daxx mRNA and protein expression are induced following exposure to type I IFNs, indicating that Daxx is an ISG [2, 3].
Here, we demonstrate that type I IFNs are powerful inhibitors of ASV replication in human and avian cells. We show that, although Daxx is upregulated by type I IFNs and essential on its own for silencing ASV gene expression in human cells, it is largely dispensable for establishment of the type I IFN-induced anti-retroviral state. Our results suggest that IFNs are capable of effectively inhibiting ASV even in the absence of Daxx, providing evidence that epigenetic silencing by Daxx is a redundant mechanism of the IFN anti-ASV response in mammalian cells.
Type I IFNs inhibit ASV replication in mammalian cells
To investigate whether type I IFNs can block the early steps in ASV replication, we treated HeLa cells with either human IFN-α or IFN-β prior to infection with an ASV-GFP reporter virus. This reporter virus is pseudotyped to express the murine leukemia virus (MuLV) amphotropic envelope protein, and is therefore capable of entry into mammalian cells. ASV-GFP contains an intact complement of replicative genes, and is fully-capable of productive infection in its natural avian host cells, but several post-transcriptional blocks in mammalian cells inhibit late events in the virus life-cycle, limiting infection to a single round in these cells [2, 3]. ASV-GFP infection of mammalian cells, however, recapitulates key early events of the retroviral life-cycle, including entry, uncoating, reverse-transcription and integration. As diminished GFP expression is a faithful readout of Daxx-dependent silencing, we have previously employed ASV-GFP to identify post-integration silencing of retroviral gene-expression as a Daxx-sensitive step [2, 3].
Type I IFNs Inhibit ASV replication in avian cells
Daxx is induced by type I IFNs in mammalian and avian cells
Daxx is not essential for type I IFN-mediated inhibition of ASV replication in mammalian cells
The two salient findings of this study are that (1) type I IFNs can potently inhibit ASV replication in mammalian and avian cells, and (2) that although Daxx is IFN-inducible, IFN-mediated anti-ASV activity in mammalian cells does not require Daxx. Together with our previous demonstration that Daxx is essential for anti-retroviral host defense in the absence of IFNs [2, 3], our current observations support a model for Daxx function in which Daxx protects against ASV (by epigenetic repression of ASV proviral gene expression) prior to induction of IFNs. Once induced, type I IFNs can establish an anti-retroviral state in which Daxx is not essential.
A Daxx ortholog has been identified in Drosophila and other insects , predating by ~150 million years the emergence of IFN-α/β genes, which can be traced back ~250 million years to the time when reptiles and birds diverged from each other [13, 14]. It is thus possible that Daxx represents an ancient, metazoan anti-retroviral protein the function of which remains essential in the absence of IFNs, but which has since been rendered redundant by the relatively-recent emergence of the type I IFN system in higher vertebrates.
Alternatively, our finding that IFN-mediated protection against ASV is Daxx-independent may be explained simply by activation of mechanistically distinct, but functionally redundant ISGs. Indeed, dependence on a single ISG may be detrimental to the host in the face of a virus infection, as viruses are capable of rapid evolution and consequent subversion of antiviral host proteins. Several viruses are known to target Daxx. For example, the human cytomegalovirus (HCMV) virion tegument protein pp71, as well as the adenovirus E1B-55 K protein have been shown to induce degradation of Daxx via the proteasome . Redundant antiviral mechanisms ensure that multiple host defense strategies are in place, should any one, e.g. Daxx, be compromised by virus infection. We speculate that in mammalian cells, IFNs target multiple early steps in the ASV life cycle upstream of where Daxx is proposed to act, including entry, capsid disassembly, uncoating, nuclear entry/reverse transcription, and integration. As IFNs are capable of anti-ASV activity even in the absence of Daxx, epigenetic repression of proviral DNA by Daxx likely represents only one of the many diverse pathways, including those activated by APOBEC3G, TRIM5, TRIM22, and MXB, which are deployed by IFNs to restrict early steps of retroviral replication [16–18]. For example, APOBEC3G triggers damaging hypermutation of retroviral cDNA following reverse transcription, TRIM5 blocks HIV-1 by inhibiting viral cDNA synthesis, and MXB has been reported to inhibit HIV-1 DNA integration [16–18]. Induction of these or similar restriction factors may account for IFN-mediated protection against ASV in the absence of Daxx.
This work was supported by an ACS Research Scholar Grant (RSG-09-195-01 MPC), and National Institutes of Health grant R21AI104212 (SB), and by a National Institutes of Health grant RO1CA71515 (RK and AMS). Additional support was provided by the W.W. Smith Charitable Trust (SB and AMS), and by the F.M. Kirby Foundation (SB). The funding bodies had no role in the design, collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.
- Yang X, Khosravi-Far R, Chang HY, Baltimore D: Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 1997, 89: 1067-1076. 10.1016/S0092-8674(00)80294-9PubMedPubMed CentralView ArticleGoogle Scholar
- Greger JG, Katz RA, Ishov AM, Maul GG, Skalka AM: The cellular protein daxx interacts with avian sarcoma virus integrase and viral DNA to repress viral transcription. J Virol 2005, 79: 4610-4618. 10.1128/JVI.79.8.4610-4618.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Shalginskikh N, Poleshko A, Skalka AM, Katz RA: Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx. J Virol 2013, 87: 2137-2150. 10.1128/JVI.02026-12PubMedPubMed CentralView ArticleGoogle Scholar
- Platanias LC: Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 2005, 5: 375-386. 10.1038/nri1604PubMedView ArticleGoogle Scholar
- Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD: How cells respond to interferons. Annu Rev Biochem 1998, 67: 227-264. 10.1146/annurev.biochem.67.1.227PubMedView ArticleGoogle Scholar
- Schaefer-Klein J, Givol I, Barsov EV, Whitcomb JM, VanBrocklin M, Foster DN, Federspiel MJ, Hughes SH: The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors. Virology 1998, 248: 305-311. 10.1006/viro.1998.9291PubMedView ArticleGoogle Scholar
- Balachandran S, Thomas E, Barber GN: A FADD-dependent innate immune mechanism in mammalian cells. Nature 2004, 432: 401-405. 10.1038/nature03124PubMedView ArticleGoogle Scholar
- Fernandez M, Porosnicu M, Markovic D, Barber GN: Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. J Virol 2002, 76: 895-904. 10.1128/JVI.76.2.895-904.2002PubMedPubMed CentralView ArticleGoogle Scholar
- Balachandran S, Barber GN: Defective translational control facilitates vesicular stomatitis virus oncolysis. Cancer Cell 2004, 5: 51-65. 10.1016/S1535-6108(03)00330-1PubMedView ArticleGoogle Scholar
- Stojdl DF, Lichty BD, enOever BR, Paterson JM, Power AT, Knowles S, Marius R, Reynard J, Poliquin L, Atkins H, Brown EG, Durbin RK, Durbin JE, Hiscott J, Bell JC: VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 2003, 4: 263-275. 10.1016/S1535-6108(03)00241-1PubMedView ArticleGoogle Scholar
- Suerth JD, Maetzig T, Galla M, Baum C, Schambach A: Self-inactivating alpharetroviral vectors with a split-packaging design. J Virol 2010, 84: 6626-6635. 10.1128/JVI.00182-10PubMedPubMed CentralView ArticleGoogle Scholar
- Santiago A, Godsey AC, Hossain J, Zhao LY, Liao D: Identification of two independent SUMO-interacting motifs in Daxx: evolutionary conservation from Drosophila to humans and their biochemical functions. Cell Cycle 2009, 8: 76-87. 10.4161/cc.8.1.7493PubMedView ArticleGoogle Scholar
- Roberts RM, Liu L, Guo Q, Leaman D, Bixby J: The evolution of the type I interferons. J Interferon Cytokine Res 1998, 18: 805-816. 10.1089/jir.1998.18.805PubMedView ArticleGoogle Scholar
- Crawford NG, Faircloth BC, McCormack JE, Brumfield RT, Winker K, Glenn TC: More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs. Biol Lett 2012, 8: 783-786. 10.1098/rsbl.2012.0331PubMedPubMed CentralView ArticleGoogle Scholar
- Schreiner S, Wodrich H: Virion factors that target Daxx to overcome intrinsic immunity. J Virol 2013, 87: 10412-10422. 10.1128/JVI.00425-13PubMedPubMed CentralView ArticleGoogle Scholar
- Malim MH, Bieniasz PD: HIV restriction factors and mechanisms of evasion. Cold Spring Harb Perspect Med 2012, 2: a006940.PubMedPubMed CentralView ArticleGoogle Scholar
- Hattlmann CJ, Kelly JN, Barr SD: TRIM22: a diverse and dynamic antiviral protein. Mol Biol Int 2012, 2012: 153415.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu Z, Pan Q, Ding S, Qian J, Xu F, Zhou J, Cen S, Guo F, Liang C: The interferon-inducible MxB protein inhibits HIV-1 infection. Cell Host Microbe 2013, 14: 398-410. 10.1016/j.chom.2013.08.015PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.