- Short report
- Open Access
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
- Innate immunity
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.
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