PML‐NB‐dependent type I interferon memory results in a restricted form of HSV latency
2021; Springer Nature; Volume: 22; Issue: 9 Linguagem: Inglês
10.15252/embr.202152547
ISSN1469-3178
AutoresJon B. Suzich, Sean R. Cuddy, Hiam Baidas, Sara A. Dochnal, Eugene Ke, Austin R. Schinlever, Aleksandra Babnis, Chris Boutell, Anna R. Cliffe,
Tópico(s)Herpesvirus Infections and Treatments
ResumoArticle1 July 2021free access Source DataTransparent process PML-NB-dependent type I interferon memory results in a restricted form of HSV latency Jon B Suzich Jon B Suzich Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Sean R Cuddy Sean R Cuddy Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Hiam Baidas Hiam Baidas Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Sara Dochnal Sara Dochnal Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Eugene Ke Eugene Ke Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Austin R Schinlever Austin R Schinlever Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Aleksandra Babnis Aleksandra Babnis Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Chris Boutell Chris Boutell MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, UK Search for more papers by this author Anna R Cliffe Corresponding Author Anna R Cliffe [email protected] orcid.org/0000-0003-1136-5171 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Jon B Suzich Jon B Suzich Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Sean R Cuddy Sean R Cuddy Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Hiam Baidas Hiam Baidas Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Sara Dochnal Sara Dochnal Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Eugene Ke Eugene Ke Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Austin R Schinlever Austin R Schinlever Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Aleksandra Babnis Aleksandra Babnis Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Chris Boutell Chris Boutell MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, UK Search for more papers by this author Anna R Cliffe Corresponding Author Anna R Cliffe [email protected] orcid.org/0000-0003-1136-5171 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Author Information Jon B Suzich1, Sean R Cuddy2, Hiam Baidas1, Sara Dochnal1, Eugene Ke1, Austin R Schinlever1, Aleksandra Babnis1, Chris Boutell3 and Anna R Cliffe *,1 1Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA 2Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, USA 3MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, UK **Corresponding author. Tel: +1 434 9247780; E-mail: [email protected] EMBO Reports (2021)22:e52547https://doi.org/10.15252/embr.202152547 See also: SK Weller & NA Deluca (September 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalize with PML-NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate. Synopsis Latent HSV-1 genomes in peripheral neurons possess a long-term memory of the IFN response in primary infection. This memory is characterized by association of latent genomes with IFN-induced PML-NBs and ultimately restriction of viral reactivation. Primary sympathetic and sensory neurons do not have detectible PML-NBs. PML- NBs form with type I IFN treatment and persist even following cessation of IFN signaling. HSV-1 genomes become entrapped by PML-NBs throughout latency only when type I IFN is present during initial infection. PML-NBs are not required for latency, but latent genomes are more restricted for reactivation when they are entrapped. Introduction Herpes simplex virus-1 (HSV-1) is a ubiquitous pathogen that persists in the form of a lifelong latent infection in the human host. HSV-1 can undergo a productive lytic infection in a variety of cell types; however, latency is restricted to post-mitotic neurons, most commonly in sensory, sympathetic, and parasympathetic ganglia of the peripheral nervous system (Baringer & Swoveland, 1973; Warren et al, 1978; Baringer & Pisani, 1994; Richter et al, 2009). During latent infection, the viral genome exists as an episome in the neuronal nucleus, and there is considerable evidence that on the population level viral lytic gene promoters assemble into repressive heterochromatin (Wang et al, 2005; Cliffe & Knipe, 2008; Cliffe et al, 2009; Kwiatkowski et al, 2009). The only region of the HSV genome that undergoes active transcription, at least in a fraction of latently infected cells, is the latency-associated transcript (LAT) locus (Stevens et al, 1987; Kramer & Coen, 1995). Successful establishment of a latent gene expression program requires a number of molecular events, likely influenced by both cellular and viral factors, and is not uniform (Efstathiou & Preston, 2005). Significant heterogeneity exists in expression patterns of both lytic and latent transcripts in latently infected neurons, as well as in the ability of latent genomes to reactivate in response to different stimuli (Sawtell, 1997; Proenca et al, 2008; Catez et al, 2012; Ma et al, 2014; Maroui et al, 2016; Nicoll et al, 2016). This heterogeneity could arise from viral genome copy number, exposure to different inflammatory environments, or intrinsic differences in the neurons themselves. Furthermore, there is growing evidence that heterogeneity in latency may ultimately be reflected in part by the association of viral genomes with different nuclear domains or cellular proteins (Catez et al, 2012; Maroui et al, 2016). However, what determines the subnuclear distribution of latent viral genomes is not known. In addition, it is currently unclear whether viral genome association with certain nuclear domains or cellular proteins results in an increased or decreased ability of the virus to undergo reactivation. The aim of this study was to determine whether the presence of interferon during initial HSV-1 infection can intersect with the latent viral genome to regulate the type of gene silencing and ultimately the ability to undergo reactivation. Because the fate of viral genomes and their ability to undergo reactivation can be readily tracked, latent HSV-1 infection of neurons also serves as an excellent system to explore how exposure to innate immune cytokines can have a lasting impact on peripheral neurons. Latent HSV-1 genomes have been shown to associate with promyelocytic leukemia nuclear bodies (PML-NBs) in mouse models of infection, as well as in human autopsy material (Catez et al, 2012; Maroui et al, 2016). PML-NBs are heterogeneous, phase-separated nuclear condensates that have been associated with the transcriptional activation of cellular genes (Wang et al, 2004; Bernardi & Pandolfi, 2007; Lallemand-Breitenbach & de The, 2010; Kim & Ahn, 2015; McFarlane et al, 2019), but also can recruit repressor proteins, including ATRX, Daxx, and Sp100, that promote transcriptional repression and inhibition of both DNA and RNA virus replication (Zhong et al, 2000b; Garrick et al, 2004; Bishop et al, 2006; Everett & Chelbi-Alix, 2007; Xu & Roizman, 2017). In the context of lytic infection of non-neuronal cells, PML-NBs have been shown to closely associate with HSV-1 genomes (Maul et al, 1996; Maul, 1998), and the HSV-1 viral regulatory protein ICP0 is known to disrupt the integrity of these structures by targeting PML and other PML-NB-associated proteins for degradation (Everett & Maul, 1994; Chelbi-Alix & de The, 1999; Boutell et al, 2002). PML-NBs entrapment of HSV-1 genomes during lytic infection of fibroblasts (Alandijany et al, 2018) is hypothesized to create a transcriptionally repressive environment for viral gene expression, as PML directly contributes to the cellular repression of ICP0-null mutant viruses (Everett et al, 2006). In the context of latency, neurons containing PML-encased latent genomes exhibit decreased expression levels of the LAT (Catez et al, 2012), suggesting that they are more transcriptionally silent than latent genomes localized to other nuclear domains and raising the question as to whether PML-NB-associated genomes are capable of undergoing reactivation. Studies have shown that replication-defective HSV genomes associated with PML-NBs are capable of derepressing following induced expression of ICP0 in fibroblasts (Everett et al, 2007; Cohen et al, 2018) and following addition of the histone deacetylase inhibitor trichostatin A (TSA) in cultured adult TG neurons (Maroui et al, 2016). However, it is not known if replication-competent viral genomes associated with PML-NBs are capable of undergoing reactivation triggered by activation of cellular signaling pathways in the absence of viral protein. PML-NBs can undergo significant changes in number, size, and localization depending on cell type, differentiation stage, and cell–cycle phase, as well as in response to cellular stress and soluble factors (Bernardi & Pandolfi, 2007; Lallemand-Breitenbach & de The, 2010). Interferon (IFN) treatment directly induces the transcription of PML, Daxx, Sp100, and other PML-NB constituents, which leads to elevated protein synthesis and a robust increase in both size and number of PML-NBs (Chelbi-Alix et al, 1995; Stadler et al, 1995; Grotzinger et al, 1996; Greger et al, 2005; Shalginskikh et al, 2013). During HSV-1 infection, type I IFNs are among the first immune effectors produced, and they have been shown to restrict HSV viral replication and spread both in vitro and in vivo through multiple pathways (Hendricks et al, 1991; Mikloska et al, 1998; Mikloska & Cunningham, 2001; Sainz & Halford, 2002; Jones et al, 2003). Type I IFNs are elevated within peripheral ganglia during HSV-1 infection (Carr et al, 1998) and have been linked with control of lytic HSV-1 replication. In an in vitro model of latency, exogenous type I IFNs also have been shown to induce neuron-specific antiviral responses that control reactivation (Linderman et al, 2017), but whether type I IFN exposure during initial infection modulates entry into latency is not known. Importantly, exposure to IFN and other cytokines has also been shown to generate innate immune memory or “trained immunity” in fibroblasts and immune cells (Kamada et al, 2018; Moorlag et al, 2018), and PML-NBs themselves are potentially important in the host innate immune response. A previous study found that the histone chaperone HIRA is re-localized to PML-NBs in response to the innate immune defenses induced by HSV-1 infection, and in this context, PML was required for the recruitment of HIRA to ISG promoters for efficient transcription (McFarlane et al, 2019). Prior exposure to type I interferons has also been shown to promote a transcriptional memory response in fibroblasts and macrophages (Kamada et al, 2018). This interferon memory leads to faster and more robust transcription of ISGs following restimulation and coincided with acquisition of certain chromatin marks and accelerated recruitment of transcription and chromatin factors (Kamada et al, 2018). Thus far, long-term memory of cytokine exposure has only been investigated in non-neuronal cells, but it is conceivable that neurons, being non-mitotic and long-lived cells, also possess unique long-term responses to prior cytokine exposure. Although in vivo models are incredibly powerful tools to investigate the contribution of the host immune response to HSV infection, they are problematic for investigating how individual components of the host’s immune response specifically regulate neuronal latency. Conversely, in vitro systems provide a simplified model that lacks many aspects of the host immune response. Therefore, to investigate the role of type I IFN on HSV-1 latency and reactivation, we utilized a model of latency in primary murine sympathetic neurons (Cliffe et al, 2015), which allowed us to manipulate conditions during initial HSV-1 infection and trigger synchronous robust reactivation. Using this model, we show that primary neurons isolated from mouse peripheral ganglia are largely devoid of detectable PML-NBs but PML-NBs form following type I IFN exposure and persist even when ISG gene expression and production of other antiviral proteins have returned to baseline. Neither exogenous type I IFN nor detectable PML-NBs are required for HSV gene silencing and entry into latency in this model system, but, importantly, the presence of IFNα specifically at the time of initial infection results in the entrapment of viral genomes in PML-NBs and a more restrictive form of latency that is less able to undergo reactivation. This study therefore demonstrates how the viral latent genome has a long-term memory of the innate response during de novo HSV infection that results in entrapment of genomes in PML-NBs and a more repressive form of latency. Results Interferon induces the formation of detectable PML-NBs in primary sympathetic and sensory neurons isolated from postnatal and adult mice We initially set out to investigate the contribution of PML-NBs to HSV latency and reactivation using primary sympathetic and sensory neurons that have been well characterized as in vitro models of HSV latency and reactivation (Wilcox & Johnson, 1987; Wilcox et al, 1990; Camarena et al, 2010; Cliffe et al, 2015; Ives & Bertke, 2017; Cuddy et al, 2020). In addition, primary neuronal systems allow for much more experimental control of specific conditions during de novo infection and can be easily manipulated either immediately prior to or following infection. Peripheral neurons were isolated from the superior cervical ganglia (SCG) or trigeminal ganglia (TG) from young (post-natal day; P1) or adult (> P28) mice and cultured for 6 days prior to staining. PML-NBs were defined as detectable punctate nuclear structures by staining for PML protein. Strikingly, we observed that both SCG and TG neurons were largely devoid of detectable PML-NBs (Fig 1A). Figure 1. Type I IFN induces the formation of PML-NBs in primary peripheral neurons A. Representative images of primary neurons isolated from superior cervical ganglia (SCG) and sensory trigeminal ganglia (TG) of postnatal (P6) and adult (P28) mice stained for PML and the neuronal marker BIII-tubulin. B. SCG and TG neurons isolated from P6 and P28 mice were treated with interferon (IFN)α (600 IU/ml) for 18 h and stained for PML and BIII-tubulin. C–F. Quantification of detectable PML puncta in P6 and P28 neurons following 18-h treatment with IFNα (150 IU/ml, 600 IU/ml), IFNγ (150 IU/ml, 500 IU/ml), and IFNλ2 (100 ng/ml, 500 ng/ml). Data information: Data represent the mean ± SEM. n = 60 cells from 3 biological replicates. Statistical comparisons were made using a one-way ANOVA with Tukey’s multiple comparison (ns not significant, ****P < 0.0001). Scale bar, 20 μm. Source data are available online for this figure. Source Data for Figure 1 [embr202152547-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint In certain cell types, the transcription of certain PML-NB-associated proteins, including PML, can be induced by either type I or type II interferon (IFN) treatment, which is correlated with an increase in PML-NB size and/or number per cell (Chelbi-Alix et al, 1995; Stadler et al, 1995). Therefore, we were interested in determining whether exposure of primary sensory or sympathetic neurons to different types of IFN resulted in PML-NB formation. Type I IFN treatment using IFN-alpha (IFNα) (Fig 1B, C–F) or IFN-beta (Fig EV1A) led to a significant induction of detectable PML-NBs in both sensory and sympathetic neurons isolated from postnatal and adult mice. Representative images of IFNα-treated neurons are shown (Fig 1B), and the number of detectable PML-NBs per neurons is quantified (Fig 1C–F). The increase in detectable PML-NBs was comparable for both 150 IU/ml and 600 IU/ml of IFNα. Type II IFN (IFNγ) led to a more variable response with a small but significant increase in detectable PML-NBs in a subpopulation of sympathetic neurons. However, IFNγ treatment of sensory neurons did not result in the formation of detectable PML-NBs. Exposure of neurons to IFN-lambda 2 (IFN-λ2), a type III IFN, did not induce the formation of detectable PML-NBs in either sympathetic or sensory neuron cultures (Figs 1C–F and EV1B and C). Therefore, PML-NBs are largely undetectable in primary sympathetic and sensory neurons but can form upon exposure to type I IFNs. Click here to expand this figure. Figure EV1. Type I IFN alters the sub-cellular localization of ATRX, Daxx, and SUMO-1 in primary peripheral neurons A. Representative images of P6 SCG neurons treated with IFNβ (150 IU/ml) and stained for PML and ATRX. B. Representative images of P6 SCG treated with IFNα (600 IU/ml), IFNγ (500 IU/ml) and IFNλ2 (500 ng/ml) and stained for PML. C. Representative images of P6 TG treated with IFNα (600 IU/ml), IFNγ (500 IU/ml), and IFNλ2 (500 ng/ml) and stained for PML. D–F. Representative images of untreated or IFNα (600 IU/ml)-treated P6 SCG neurons stained for PML and ATRX (D), Daxx and ATRX (E), and PML and SUMO-1 (F). P6 dermal fibroblasts (DF) isolated from the same mice were used as a non-neuronal control (D–F). Data information: Scale bars, 20 μm. Download figure Download PowerPoint The absence of detectable PML-NBs in untreated primary neurons prompted us to investigate other known components of PML-NBs. We were particularly interested in ATRX and Daxx because like PML they have previously been found to be involved in restricting HSV lytic replication in non-neuronal cells (Lukashchuk & Everett, 2010; Alandijany et al, 2018; Cabral et al, 2018; McFarlane et al, 2019). Therefore, we investigated the localization of ATRX and Daxx in primary peripheral neurons. ATRX is a multifunctional, heterochromatin-associated protein that is localized to PML-NBs in human and mouse mitotic cells and is largely characterized as interacting with the Daxx histone chaperone (Lewis et al, 2010; Clynes et al, 2013). In untreated neurons, we observed abundant ATRX staining throughout the nucleus in regions that also stained strongly with Hoechst (Fig EV1D and E). This potential colocalization of ATRX with regions of dense chromatin is consistent with a previous study demonstrating that in neurons ATRX binds certain regions of the cellular genome associated with the constitutive heterochromatin modification H3K9me3 (Noh et al, 2015). Importantly, this distribution of ATRX differs from what is seen in murine dermal fibroblasts (Fig EV1D and E) and other non-neuronal cells, where there is a high degree of colocalization between ATRX and PML (Alandijany et al, 2018). Following treatment with IFNα, we found a redistribution of ATRX staining and colocalization between ATRX and the formed PML-NBs, but the majority of ATRX staining remained outside the context of PML-NBs (Fig EV1D and E). Similar to PML, sympathetic SCG and sensory TG neurons isolated from both postnatal and adult mice were devoid of detectable puncta of Daxx staining (Fig EV1E), and we did not observe extensive Daxx staining in untreated neurons as we did for ATRX. We were unable to directly co-stain for Daxx and PML; however, treatment of neurons with IFNα did induce punctate Daxx staining that strongly colocalized with puncta of ATRX (Fig EV1E), which given our previous observation of ATRX colocalization with PML following type I IFN treatment we used as a correlate for PML-NBs. We were also interested in SUMO-1, which has been shown to be required for formation of PML-NBs (Zhong et al, 2000a). Similar to ATRX and Daxx, treatment of neurons with IFNα induced punctate SUMO-1 staining in P6 SCG neurons that colocalized with PML puncta (Fig EV1F). Therefore, PML-NBs containing their well-characterized associated proteins are not detected in cultured primary neurons but form in response to type I IFN exposure. Type I IFN treatment specifically at time of infection restricts reactivation of HSV-1 from primary sympathetic neurons without affecting initial infectivity or LAT expression Because we observed that primary SCG neurons are largely devoid of PML-NBs and that PML-NBs form upon treatment with type I IFN treatment, we first wanted to clarify that latency was maintained in the absence of IFN and presumably without PML-NB formation, consistent with our previous data (Cuddy et al, 2020). SCG neurons were infected at a multiplicity of infection (MOI) of 7.5 plaque forming units (PFU)/cell with HSV-1 Us11-GFP presence of acyclovir (ACV). The ACV was removed after 6 days, and the neuronal cultures were monitored to ensure the no GFP-positive neurons were present (Fig 2A). We found that latency could be established and maintained for up to 5 days following removal of ACV (Fig 2B). Reactivation was triggered by PI3K inhibition using LY294002, as previously described (Camarena et al, 2010; Kim et al, 2012; Kobayashi et al, 2012; Cliffe et al, 2015), and quantified based on the number of Us11-GFP neurons in the presence of WAY-150138 which blocks packaging of progeny genomes and thus cell-to-cell spread (van Zeijl et al, 2000). These data therefore indicate that exogenous IFN is not required to induce a latent state in this model system. Figure 2. Type I IFN treatment solely at time of infection inhibits LY294002-mediated reactivation of HSV-1 in primary sympathetic SCG neurons Schematic of the primary postnatal sympathetic neuron-derived model of HSV-1 latency. Reactivation from latency is quantified by Us11-GFP expressing neurons following addition of the PI3K inhibitor LY294002 (20 μM) in the presence of WAY-150168, which prevents cell-to-cell spread. The arrow indicates the time of LY294002 treatment at 5 days post-establishment of latency. n = 9 biological replicates. Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures infected with HSV-1 in the presence or absence of IFNα (600 IU/ml), then treated with an α-ΙFNAR1 neutralizing antibody. n = 9 biological replicates. RT–qPCR for viral mRNA transcripts at 3 days post-LY294002-induced reactivation of SCGs infected with HSV-1 in the presence or absence of IFNα. n = 9 biological replicates. RT–qPCR for viral mRNA transcripts at 20 h post-LY294002-induced reactivation in SCGs infected with HSV-1 in the presence of absence of IFNα. n = 9 biological replicates. Relative amount of viral DNA at time of reactivation (8 dpi) in SCG neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n = 9 biological replicates. Quantification of vDNA foci detected by click chemistry at time of reactivation (8 dpi) in SCG neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n = 60 genomes from 3 biological replicates. LAT mRNA expression at time of reactivation (8 dpi) in neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n = 9 biological replicates. Data information: Data represent the mean ± SEM. Statistical comparisons were made using a Mann–Whitney test (ns not significant, **P < 0.01, ****P < 0.0001). Source data are available online for this figure. Source Data for Figure 2 [embr202152547-sup-0003-SDataFig2.xlsx] Download figure Download PowerPoint We next turned our attention to whether type I IFN treatment at the time of infection impacted the ability of HSV to establish latency or reactivate in this model system. SCG neurons were pretreated with IFNα (600 IU/ml) for 18 h and during the initial 2 h HSV inoculation. Following inoculation, IFNα was washed out and an IFNAR1 blocking antibody was used to prevent subsequent type I IFN signaling through the receptor. To confirm the effectiveness of the IFNAR1 ab to block detectable IFN signaling, we validated it by its ability to block ISG expression (ISG15) in cultured SCG neurons by RT–qPCR (Fig EV2A). Reactivation was induced and initially quantified based on the number of GFP-positive neurons at 3 days post-stimuli. We found that full reactivation was restricted in neurons exposed to type I IFN just prior to and during de novo infection (Fig 2C). We further confirmed this IFNα-mediated restriction of latency by the induction of lytic mRNAs upon reactivation. IFNα treatment at the time of infection significantly decreased the expression of immediate-early gene (ICP27), early gene (ICP8), and late gene (gC) at 3 days post-reactivation (Figs 2D and EV2AB and C). There were very few GFP-positive neurons and little to no viral gene expression in mock reactivated controls, further indicating that latency can be established in the presence and absence of IFN. Click here to expand this figure. Figure EV2. Type I IFN treatment solely at time of infection inhibits LY294002-mediated reactivation of HSV-1 in primary sympathetic SCG neurons A. RT–qPCR for ISG15 mRNA expression in SCG neurons treated with IFNα (600 IU/ml) in the presence or absence of anti-mouse IFNAR1 antibody (1:1,000). n = 6 biological replicates. B, C. RT–qPCR for viral mRNA transcripts at 3 days post-LY294002-induced reactivation of SCGs infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n = 9 biological replicates. D, E. RT–qPCR for viral mRNA transcripts at 20 h post-LY294002-induced reactivation in SCGs infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n = 9 biological replicates. Data information: Data represent the mean ± SEM. Statistical comparisons were made using a one-way ANOVA with Tukey’s multiple comparison (A) or a Mann–Whitney test (ns not significant, ***P < 0.001, ****P < 0.0001). Download figure Download PowerPoint Reactivation of HSV in this system proceeds over two phases. GFP-positive neurons is a readout for full reactivation or Phase II. However, we and others have observed an initial wave of lytic gene expression that occurs prior to and independently of viral DNA replication at around 20 h post-stimulus, termed Phase I (Du et al, 2011; Kim et al, 2012; Cliffe et al, 2015; Cliffe & Wilson, 2017). Therefore, to determine whether IFNα treatment at the time of infection restricted the Phase I wave of lytic, we carried out RT–qPCR to detect representative immediate-early (ICP27), early (ICP8), and late (gC) transcripts at 20 h post-addition of LY294002. We found significantly decreased expression in the IFNα-treated neurons (Figs 2E and EV2D and E). This is interesting as exogenous type I IFNs have previously been shown to suppress reactivation in murine neurons by preventing Phase I and are rendered ineffective once Phase I viral products accumulate (Linderman et al, 2017). Therefore, type I IFN treatment solely at the time of infection has a long-term effect on the ability of HSV to initiate lytic gene expression and undergo reactivation. Because IFN treatment could reduce nuclear trafficking of viral capsids during initial infection or impact infection efficiency, we next determined whether equivalent numbers of viral genomes were present in the neuronal cultures. At 8 dpi, we measured relative viral DNA genome copy numbers in SCG neurons that were treated with IFNα compared to untreated controls and found no significant difference (Fig 2F). To further confirm that equivalent genomes were present in the neuronal nuclei, we infected neurons with HSV-1-containing EdC-incorporat
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