Rapid recruitment of host immune regulators to vDNA upon nuclear entry is crucial for the efficient induction of host immunity and suppression of viral replication. Pre-existing restriction factors, as part of the intrinsic immune response, directly induce viral genome silencing (Yan and Chen 2012, Tavalai and Stamminger 2009). PRRs, as part of the innate immune response, activate IFN-signaling cascades leading to the expression of ISGs (Orzalli and Knipe 2014, Hannoun et al. 2016). It remains unclear whether recruitment of the intrinsic and innate immune factors to vDNA occurs simultaneously or sequentially because of the technical difficulties associated with viral genome detection under low MOI conditions. Utilizing click chemistry (Salic and Mitchison 2008, Chehrehasa et al. 2009), we provided a non-invasive assay to quantitatively assess the recruitment efficiency of host immune factors to EdU-labeled vDNA under low MOI conditions (MOI of ≤ 3 PFU/cell within 90 mpi). This protocol allowed direct visualization of vDNA (Figure 11) and enabled detailed investigation into the temporal recruitment of intrinsic and innate immune factors to infecting viral genomes.
PML-NB constituent proteins (PML, SP100, Daxx, ATRX, PIAS-1, and MORC3) are the most prominent examples of host cell restriction factors (Everett et al. 2008, Everett et al. 2006, Lukashchuk and Everett 2010, Brown et al. 2016, Sloan et al. 2016). It was initially reported that PML-NBs disappear during HSV-1 infection as a consequence of ICP0 expression (Maul and Everett 1994). ΔICP0 HSV-1 was very valuable to understand the relationship between PML-NBs and HSV-1 during lytic and latent infections (Stow and Stow 1986, Sacks and Schaffer 1987). Plaque-edge recruitment assays have shown that PML-NB proteins are recruited to sites associated with incoming viral genomes (Everett et al. 2004a). These recruitment phenotypes correlated with suppression of viral gene transcription. The presence of PML, the major scaffolding protein of PML-NBs, is not necessary for recruitment of other PML-NB proteins (e.g., SP100 and Daxx) to vDNA. Consistently, PML-NB proteins (SP100 and Daxx) independently and additively promote transcriptional repression of viral genes as demonstrated by
114 co-depletion and triple-depletion studies (Lukashchuk and Everett 2010, Everett et al. 2006, Everett et al. 2008, Brown et al. 2016, Glass and Everett 2013). The antiviral role of PML-NBs against HSV-1 extends to latent infection. Indeed, the viral genomes were shown to be stably entrapped within PML-NBs in latently- infected mouse neurons and quiescently-infected cell culture (Catez et al. 2012, Maroui et al. 2016, Everett et al. 2007). The microscopy observations in this study demonstrated for the first time that the viral genomes are rapidly and stably entrapped within PML-NBs immediately upon genome delivery to the nucleus during lytic infection (Figure 12). HSV-1 expresses ICP0 which localizes to PML-NBs to induce PML degradation leading to the dispersal of PML-NBs. This process disrupts the spatial recruitment of PML-NB restriction factors to vDNA, and allows the virus to counteract PML-NB mediated viral genome silencing and initiate lytic replication (Figure 15, 16, 17, and 18) (Maul and Everett 1994, Chelbi-Alix and de The 1999, Boutell et al. 2002, Boutell et al. 2011). In the absence of ICP0 and under MOI conditions that do not saturate intrinsic immunity, the viral genomes remained stably silenced and entrapped within PML-NBs. Importantly, this process occurred in the absence of ISG expression, demonstrating that vDNA entry into the nucleus alone is not sufficient for induction of innate immune response (Figure 24 and 25). In support of this conclusion, no stable localization between PRR IFI16 (a key regulator of host innate immunity) and vDNA was observed prior to the expression of viral genes (Figure 19) (Unterholzner et al. 2010, Orzalli et al. 2012). Live cell microscopy demonstrated that IFI16 puncta were transiently formed on the nuclear periphery of infected cells (Everett 2015, Diner et al. 2016). This observation raised the speculation that IFI16 is rapidly recruited to vDNA upon nuclear entry leading to the induction of ISG expression (Orzalli et al. 2012, Orzalli et al. 2016). Yet, no direct or indirect detection of vDNA was demonstrated in these studies (Everett 2015, Diner et al. 2016). Moreover, the number of IFI16 puncta observed did not correlate with the viral genome copy number expected to be delivered to the nucleus at high MOI (MOI of ≥ 10 PFU/cell) (Everett 2015, Diner et al. 2016). Although the interaction between IFI16 and vDNA have been previously demonstrated by ChIP (Johnson et al. 2014), direct detection of EdU- labeled vDNA did not reveal any significant colocalization between IFI16 and vDNA under low MOI conditions (Figure 19). However, this experiment was
115 conducted at fixed time points post-infection and does not exclude the possibility of transient interactions between IFI16 and vDNA.
Previous plaque-edge recruitment studies suggested that IFI16, similar to PML-NB proteins, is stably recruited to infecting ICP4 dot-like complexes that contain vDNA (Cuchet-Lourenco et al. 2013, Orzalli et al. 2013). Although controversial, IFI16 depletion has been shown to negatively influence the frequency of PML-NB protein recruitment to incoming viral genomes (Orzalli et al. 2013, Cuchet-Lourenco et al. 2013). Importantly, these studies have relied on the use of ICP4 signals as a proxy for viral genome localization; a suboptimal approach giving that ICP4 signals are only localized to a subset of transcriptionally active viral genome foci (Everett and Murray 2005). Consistence with these reports, we observed stable recruitment of IFI16 and PML to vDNA in newly infected cells next to developing plaques. However, IFI16 recruitment was observed only at the viral genomes expressing ICP4 while PML was recruited to vDNA irrespective of ICP4 expression (Figure 23) (Alandijany et al. 2018). These data demonstrate that the onset of viral gene expression or initiation of vDNA replication is required for IFI16-mediated sensing of HSV-1 DNA. Collectively, our data put a clear temporal context for the stable recruitment of PML and IFI16 to vDNA which correlates with the nuclear delivery of viral genome and expression of viral proteins, respectively.
In summary, the data presented in this chapter shed light on the temporal regulation of intrinsic and innate immune responses during intracellular restriction of HSV-1 infection. PML-NB associated restriction factors are rapidly recruited to vDNA upon nuclear entry leading to viral genome silencing. Saturation of or escape from this intrinsic immune response is required to induce innate immunity. Induction of viral gene transcription and/or initiation of vDNA replication triggers the stable recruitment of PRR IFI16 to viral genome foci and robust induction of ISG expression. However, HSV-1 expresses ICP0 which counteracts both intrinsic and innate immune responses by targeting PML and IFI16 for proteasome-dependent degradation.
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5. Intrinsic and innate antiviral responses are temporally and