pUL36 de-ubiquitinase activity augments both the initiation and progression of lytic virus infection in IFN–primed cells

The conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. Here, we show that the pUL36 DUB activity was required to overcome interferon-(IFN)-mediated suppression of both plaque initiation and progression to productive infection. Immediately upon virus entry, incoming tegument-derived pUL36-DUB activity helped the virus to escape intrinsic antiviral resistance and efficiently initiate lytic virus replication in IFN-primed cells. Subsequently, de novo expressed pUL36-DUB augmented the efficiency of productive infection and virus yield. Interestingly, removal of IFN shortly after inoculation only resulted in a partial rescue of plaque formation, indicating that an IFN-induced defense mechanism eliminates invading virus particles unless counteracted by pUL36-DUB activity. Taken together, we demonstrated that the pUL36 DUB disarms IFN-induced antiviral responses at two levels, namely, to protect the infectivity of invading virus as well as to augment productive virus replication in IFN-primed cells. Author Summary HSV-1 is an ubiquitous human pathogen that is responsible for common cold sores but may also cause life-threatening disease. pUL36 is an essential and conserved protein of infectious herpesvirus virions with a unique de-ubiquitinating (DUB) activity. The pUL36 DUB is dispensable for efficient virus infection in cell culture but represents an important viral immune evasin in vivo. Here, we showed that tegument-derived DUB activity delivered by the invading virus particles is required to overcome IFN-induced host resistance and to initiate efficient lytic infection. De novo expressed pUL36 DUB subsequently augments productive infection and virus yield. These data indicate that the pUL36 DUB antagonizes the activity of yet unidentified IFN-inducible E3 ligases to facilitate productive infection at multiple levels. Our findings underscore the therapeutic potential of targeting conserved herpesvirus DUBs to prevent or treat herpesvirus disease.

assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell 23 culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. 24 Here, we show that the pUL36 DUB activity was required to overcome interferon-(IFN)-mediated 25 suppression of both plaque initiation and progression to productive infection. Immediately upon 26 virus entry, incoming tegument-derived pUL36-DUB activity helped the virus to escape intrinsic 27 antiviral resistance and efficiently initiate lytic virus replication in IFN-primed cells. Subsequently, 28 de novo expressed pUL36-DUB augmented the efficiency of productive infection and virus yield. 29 Interestingly, removal of IFN shortly after inoculation only resulted in a partial rescue of plaque 30 formation, indicating that an IFN-induced defense mechanism eliminates invading virus particles 31 unless counteracted by pUL36-DUB activity. Taken together, we demonstrated that the pUL36 32 DUB disarms IFN-induced antiviral responses at two levels, namely, to protect the infectivity of 33 invading virus as well as to augment productive virus replication in IFN-primed cells.
Introduction 46 To limit and suppress viral infection, cells have evolved a rich arsenal of restriction factors, which 47 collectively form the innate antiviral immune system. Inherent mechanisms that operate in cells 48 without stimulation, referred to as intrinsic immunity, include factors that prevent (e.g. TRIM-5α), 49 sense (e.g. RIG-I, TLR3), or suppress the progression of infection (e.g. PML or PKR). The expression 50 levels of intrinsic effectors substantially vary between different cell types and tissues but can be 51 stimulated by the activity of interferons (IFNs) (1). Such genes are referred to as IFN-stimulated 52 genes (ISGs). When cells sense infection via recognition of viral pathogen-associated molecular 53 patterns (PAMPs) through pattern recognition receptors (PRRs), they activate signaling cascades 54 culminating in the IRF3-and NF-κB-mediated induction and secretion of type I IFNs (1-3). Through 55 autocrine and paracrine activation of the type I IFN receptor (IFNAR), the expression of several 56 hundred ISGs is stimulated (4-6). The production of ISGs in turn installs an antiviral state in both 57 infected as well as neighboring, uninfected cells, which subsequently hampers both susceptibility 58 as well as permissiveness to productive infection. 59 Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus that productively infects a 60 large variety of cell types. IFN-induced effector mechanisms inhibit HSV-1 at various stages of its 61 life cycle (7,8) including cell entry (9), initiation of IE gene expression (10-18) and efficient viral 62 protein production (19). To counteract these antiviral effectors, HSV-1 has evolved a plethora of 63 viral immune evasins that efficiently interfere with the innate immune system during lytic 64 infection (reviewed in (20)). Many of the host's innate defense mechanisms depend on cycles of 65 ubiquitination/de-ubiquitination of target proteins to modulate their function (reviewed in (21, 5 66 22)). In turn, HSV-1 usurps the ubiquitin system to counter these defenses through the functions 67 of the viral proteins ICP0 (reviewed in (23)) and pUL36 (see below). 68 Herein, we characterized the function of the essential large tegument protein pUL36 (also named 69 VP1-2) in the context of an established innate immune response. pUL36 is well-conserved among 70 the different herpesviruses and contributes multiple indispensable functions both early 71 (tegument-associated) and late (free and tegument-associated) in the herpesvirus life cycle (24-72 29). Upon entry of a virus particle into a cell, pUL36 is retained on the incoming capsid to facilitate 73 microtubule-mediated capsid transport towards the nucleus and docking at the nuclear pores (24, 74 26, 29-36). Late in infection, the pUL36 protein is essential for secondary envelopment and 75 tegument assembly (24,27,29,34). Besides its essential, structural functions, pUL36 and its 76 homologs also comprise an N-terminal de-ubiquitinating enzymatic activity (DUB) (37, 38) which, 77 despite its conservation, is not essential for HSV-1 replication in vitro (39). Interestingly, an N-78 terminal pUL36 fragment, which comprises the catalytically-active DUB domain, can be detected 79 in infected cells at late times of infection (38). On the molecular level, the DUB is a cysteine 80 protease that can cleave both K48-and K63-linked poly-ubiquitin chains (38, 39). As such DUB 81 activity maintains pUL36 stability by de-ubiquitinating itself (39). In other herpesviruses, the DUB 82 homologs fulfil a variety of different functions ranging from antagonizing the innate immune and 83 DNA damage responses, facilitating appropriate intracellular capsid motility and tegument 84 assembly, and augmenting infection both in vitro and in vivo (40-51). 85 Since ubiquitination/de-ubiquitination cycles are integral to innate immunity signaling, the HSV-1 86 pUL36 DUB might also interfere with innate immunity at multiple levels: (i) IFN induction, (ii) IFN 87 signaling (after IFNAR activation) and (iii) ISG functions. Indeed, the pUL36 DUB actively 88 contributes to reducing the induction of type I IFNs by reversing the ubiquitination of TRAF3 (49) 89 and STING (50), and pUL36 inhibits type I IFN signaling by binding to the IFNAR independently of 90 its DUB activity (52). 91 However, its functions during an established IFN response have not been characterized so far. 92 Herein, we show that the HSV-1 pUL36 DUB is required to disarm IFN-induced antiviral effector 93 mechanisms that interfere with the induction of lytic infection at single cell level. These effects 94 were mediated by tegument-associated DUB activity. Moreover, we demonstrate that the DUB 95 was also required later in infection for efficient production of progeny virus in IFN-treated cells. 96 Collectively, our data highlight an important role of the pUL36 DUB in antagonizing the IFN-97 induced and ubiquitination-mediated cellular defense against HSV-1 both very early, in its 98 tegument-associated form, and late in infection.

101
Comparison of replication characteristics of parental and pUL36.C65A HSV-1 strains

117
At later time points, IFN-α pre-treatment alone reduced virus yield of the DUB mutant by 10-fold, which 118 increased to over 100-fold upon combined IFN treatment (Fig. 1A). Accordingly, we observed with IFN-α 119 alone a 30%, and with IFN-α in combination with IFN-γ a 70% reduction in plaque size for the DUB mutant 120 in comparison to its parental HSV-1 KOS strain (Fig. 1B). Of note, we also noted a modest, but nevertheless 121 reproducible reduction in plaque numbers for the DUB mutants compared to their respective parental 122 strains (c.f. below).

123
We then asked whether the drop in yield after multi-cycle replication and the associated reduction in 124 plaque size of the DUB mutant were due to a reduced production of infectious virions per cell, or due to a 125 delay in productive infection. We thus performed high MOI infection, and analyzed the yield at 24 hpi from 126 Vero cells using an MOI of 10 for untreated cells, an MOI of 20 for IFN-α, and an MOI of 100 for cells pre-127 treated with both IFN-α and IFN-γ ( Fig S2) (50)) and HSV1 (KOS) (described earlier 144 (39)). For both HSV-1 parental strains, the IFN-α induction with 500 IU/ml decreased the number of 145 plaques by about 1.5-to 2-fold ( Fig. 2A), or in other words, nearly doubled the particle-to-pfu ratio, as 146 both the mock and the IFN-treated cells were inoculated with the same virus dose. Moreover, more 147 extensive IFN treatment (500 IU/ml IFN-α + 100 IU/ml IFN-γ vs. 500 IU/ml IFN-α vs. 100 IU/ml IFN-α) lead 148 to a stronger repression of plaque formation (Fig. S3). Importantly, IFN-induction reduced plaque 149 formation of both HSV-1 strain (17 + and KOS) DUB mutants significantly stronger than that of their 150 respective parental strains (p < 0.01 for both DUB mutants), consistent with a further increase in the 151 particle-to-pfu ratio by about two-fold ( Fig. 2A and S3A, C). This effect was evident both at low MOI (i.e. 152 100-200 PFU per 6-well) and at high MOI using serially diluted inocula (Fig. S4). Immunolabeling for the 153 immediate early HSV-1 protein ICP4 at 24 hpi ensured that even very small foci and plaques consisting of 154 few infected cells were not missed.

155
While we had initially analyzed the pUL36-DUB mutants in Vero cells, we next asked whether they were 156 also impaired in the initiation of plaque formation in human SK-N-SH, RPE-1, and HaCaT cells. Consistent 157 with our previous results, the DUB mutants generated significantly fewer plaques in human cells after pre-158 treatment with IFN-α (Fig. 2B (Fig. 4A, left).  for the HSV1(17 + )Lox-CheVP26 BAC harboring the mCherry-VP26 fusion (29). The C65A equivalent 283 mutation in UL36 was inserted by en passant mutagenesis (74) using the primers listed in Table   284 1. Briefly, a kanamycin resistance cassette flanked by two I-SceI restriction sites was lifted from 285 the pEP-Kan-S vector (75)

596
(A) Vero cells, either untreated or treated with 100 or 500 IU/ml IFN-α prior to inoculation only ('pre') or treated with 597 100 or 500 IU/ml IFN-α prior to and after inoculation ('pre + post'), were infected with ~100 PFU/well of KOS bacmid-598 derived parental or DUB mutant viruses. (B) As in A, but here 500 IU/ml IFN-α treatment applied throughout the 599 whole assay ('pre + post') was compared to treatment with 500 IU/ml IFN-α applied only after inoculation ('post').