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.

Herpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons. This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS–STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Influenza A viruses (IAVs) have a significant impact on public health through seasonal epidemics. Moreover, the sporadic emergence of zoonotic and pandemic strains represents an additional global threat. Understanding how novel IAV strains adapt to infect and transmit between humans is essential for effective surveillance and may provide rationale for anti-viral therapeutics. IAVs replicate their RNA genomes in the host cell nucleus. Host proteins, such as the proviral factor ANP32, are co-opted to support viral replication and transcription, whilst other inhibitory factors act to restrict. Ultimately the outcome of infection in different species depends on compatibility with these factors, however the precise nuclear localisation of these interactions is not clearly defined. It is not known whether viral RNA synthesis takes place at discrete sites, or whether the virus modifies the cell to produce viral factories in a similar manner to other viruses. We are utilising a click chemistry approach to investigate the spatial details of IAV replication and to probe the differences in nuclear localisation between human-adapted vs poorly-adapted avian origin IAVs, to gain insight into mechanisms of host restriction. Influenza genomes labelled with the click reagent 5-ethynyl uridine (5-EU) remain infectious and can be visualised through the cycloaddition of azide-tagged fluorescent dyes. Utilising this tool, the co-localisation of incoming viral particles with nuclear sub-domains, as well as key host factors, are being examined. Moreover, the localisation of human- vs avian-adapted virus in human and avian cells, as well as cell lines lacking key host factors, are being compared.

Mutations disrupting the Toll-like receptor 3 (TLR3)–dependent–interferon pathway can underlie herpes simplex encephalitis (HSE) of childhood caused by herpes simplex virus 1 (HSV1) infection. These otherwise healthy patients with HSE carry germline mutations in the TLR3-interferon circuit, including TIR domain–containing adapter–inducing IFN-β (TRIF) and TANK-binding kinase 1 (TBK1).1Sancho-Shimizu V. Pérez de Diego R. Lorenzo L. Halwani R. Alangari A. Israelsson E. et al.Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency.J Clin Invest. 2011; 121: 4889-4902Crossref PubMed Scopus (178) Google Scholar, 2Herman M. Ciancanelli M. Ou Y.-H. Lorenzo L. Klaudel-Dreszler M. Pauwels E. et al.Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood.J Exp Med. 2012; 209: 1567-1582Crossref PubMed Scopus (138) Google Scholar Their dermal fibroblasts show impaired interferon production after HSV1 infection and polyinosinic-polycytidylic acid (poly[I:C]) stimulation. A number of these genes (TLR3, TRIF, and TBK1) have also been implicated in the process of autophagy. On the other hand, HSV1 is known to antagonize the antiviral interferon pathway and the autophagy machinery in part through TBK1. Specifically, TBK1 is targeted by the virus-encoded proteins ICP34.5, ICP27, VP24, and UL46, compromising antiviral interferon signaling.3Verpooten D. Yijie M. Hou S. Yan Z. He B. Control of TANK-binding kinase 1-mediated signaling by the gamma(1)34.5 protein of herpes simplex virus 1.J Biol Chem. 2009; 284: 1097-1105Crossref PubMed Scopus (156) Google Scholar, 4Zheng C. Evasion of cytosolic DNA-stimulated innate immune responses by HSV-1.J Virol. 2018; 92Crossref Scopus (51) Google Scholar In the context of autophagy, TBK1 has been reported to phosphorylate autophagy receptors, such as p62, to promote clearance of intracellular pathogens, including HSV1 in vitro.5Sparrer K.M.J. Gableske S. Zurenski M.A. Parker Z.M. Full F. Baumgart G.J. et al.TRIM23 mediates virus-induced autophagy via activation of TBK1.Nat Microbiol. 2017; 2: 1543-1557Crossref PubMed Scopus (118) Google Scholar Here we study the role of autophagy in HSV1 infection using dermal fibroblasts from healthy controls and HSE patients with autosomal dominant TBK1 (p.G159A/WT) and autosomal recessive TRIF (p.R141X/R141X) deficiencies. Despite showing normal autophagy activation after rapamycin and poly(I:C) stimulation, TBK1+/− fibroblasts showed no induction of autophagy after multiple stimuli: cyclic diguanylate monophosphate (c-di-GMP), HSV1 60mer–double-stranded DNA (dsDNA; 60mer-dsDNA), and HSV1 infection. After rapamycin, LC3B (microtubule-associated protein 1 light chain-3B) punctate signal increased by 3-fold in both control (media, 20.0%; rapamycin, 72.3%) and TRIF−/− (media, 21.8%; rapamycin, 70.0%) fibroblasts and by 6-fold (media, 11.0%; rapamycin, 61.1%) in TBK1+/− fibroblasts, suggesting that TRIF and TBK1 were not required for rapamycin-induced autophagy (Fig 1, A and B). To assess autophagy induced by means of TLR3, poly(I:C) was used to stimulate fibroblasts, leading to a 12-fold (media, 7.1%; poly[I:C], 86.5%) increase in LC3B puncta in control fibroblasts. TRIF−/− fibroblasts were unable to induce LC3B puncta, implicating TRIF in poly(I:C)-induced autophagy. However, TBK1+/− fibroblasts showed a moderate 8-fold (media, 4.0%; poly[I:C], 31.3%) induction of autophagy, suggesting its partial role in poly(I:C)-induced autophagy consistent with its partial impairment of poly(I:C)-induced interferon production (Fig 1, A and C).2Herman M. Ciancanelli M. Ou Y.-H. Lorenzo L. Klaudel-Dreszler M. Pauwels E. et al.Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood.J Exp Med. 2012; 209: 1567-1582Crossref PubMed Scopus (138) Google Scholar Although the role of the dsRNA TLR3 pathway in regulating autophagy has been documented in other cell lines, its involvement in infection remains elusive. In addition to TLR3-interferon signaling, TBK1 is also involved in the HSV1 DNA recognition pathway through stimulator of interferon genes (STING)–TBK1–interferon regulatory factor 3 (IRF3), which serves to activate type I interferons, and STING-dependent autophagy.6Burdette D.L. Monroe K.M. Sotelo-Troha K. Iwig J.S. Eckert B. Hyodo M. et al.STING is a direct innate immune sensor of cyclic di-GMP.Nature. 2011; 478: 515-518Crossref PubMed Scopus (1015) Google Scholar, 7Rasmussen S.B. Horan K.A. Holm C.K. Stranks A.J. Mettenleiter T.C. Simon A.K. et al.Activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral DNA through a mechanism dependent on stimulator of IFN genes.J Immunol. 2011; 187: 5268-5276Crossref PubMed Scopus (92) Google Scholar Fibroblasts were transfected with c-di-GMP and 60mer-dsDNA, which are known to stimulate STING-induced autophagy and interferon production, to evaluate induction of autophagy through this pathway.6Burdette D.L. Monroe K.M. Sotelo-Troha K. Iwig J.S. Eckert B. Hyodo M. et al.STING is a direct innate immune sensor of cyclic di-GMP.Nature. 2011; 478: 515-518Crossref PubMed Scopus (1015) Google Scholar, 7Rasmussen S.B. Horan K.A. Holm C.K. Stranks A.J. Mettenleiter T.C. Simon A.K. et al.Activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral DNA through a mechanism dependent on stimulator of IFN genes.J Immunol. 2011; 187: 5268-5276Crossref PubMed Scopus (92) Google Scholar Although mock-treated fibroblasts did not show significant LC3B induction, we observed a 2-fold (c-di-GMP control, 20.7%; c-di-GMP, 45.2%) and 1.8-fold (c-di-GMP control, 18.5%; c-di-GMP, 33.6%) increase in punctate LC3B in control and TRIF−/− fibroblasts, respectively, when transfected with c-di-GMP compared with the c-di-GMP control. Significant induction of LC3B puncta after 60mer-dsDNA transfection was also observed in control and TRIF−/− fibroblasts by 3-fold (L, 29.0%; 60mer-dsDNA, 84.8%) and 2-fold (L, 32.4%; 60mer-dsDNA, 67.6%), respectively. However, c-di-GMP and 60mer-dsDNA stimulation did not induce LC3B puncta in TBK1+/− fibroblasts (Fig 1, A, D, and E), suggesting TBK1 is essential for dsDNA-induced autophagy. In control fibroblasts, HSV1 infection triggered a 10-fold (noninfected, 0.2 AU; infected multiplicity of infection [MOI] 5, 1.8 AU) increase in LC3BII:I ratio and a 2-fold (noninfected, 1.0 AU; infected MOI 5, 0.5 AU) reduction in p62 protein, indicating activation of autophagy, as assessed by means of Western blotting (Fig 1, F). TRIF−/− fibroblasts also showed a 6-fold (noninfected, 0.2 AU; infected MOI 5, 1.2 AU) LC3BII:I increase after infection at MOI 5. However, TBK1+/− fibroblasts showed no change in LC3BII:I or p62 after HSV1 infection, suggesting impaired HSV1-induced autophagy. Depletion of endogenous TBK1 using small interfering RNA (siRNA) in control fibroblasts showed similar impairment of HSV1-induced autophagy (Fig 1, G). Using immunofluorescence imaging, we found that HSV1 infection triggers 2 LC3B phenotypes in control fibroblasts: perinuclear LC3B puncta in infected cells and cytoplasmic LC3B puncta in antigen-negative plaque-neighboring (“antigen-negative”) cells (Fig 2, A). Although the former occurs later in infection and is likely the phenomenon termed nuclear envelope-derived autophagy because it also stained with LC3A (Fig 2, A),8Radtke K. English L. Rondeau C. Leib D. Lippe R. Desjardins M. Inhibition of the host translation shutoff response by herpes simplex virus 1 triggers nuclear envelope-derived autophagy.J Virol. 2013; 87: 3990-3997Crossref PubMed Scopus (28) Google Scholar cytoplasmic LC3B formed early in infection (up to 3 hours after infection; Fig 2, B). Strikingly, TBK1+/− fibroblasts did not form cytoplasmic LC3B puncta in antigen-negative cells, despite being able to form perinuclear LC3B later in infection (Fig 2, A and B). Furthermore, inhibiting TBK1 in control fibroblasts using BX795 resulted in significant reduction in cytoplasmic LC3B formation (see Fig E1 and this article’s Methods section in the Online Repository at www.jacionline.org). Although the lack of early autophagic induction was specific to TBK1+/− fibroblasts, TRIF−/− fibroblasts only showed delayed induction of autophagy (see Fig E2 in this article's Online Repository at www.jacionline.org), suggesting its partial involvement in HSV1-induced autophagy. Antigen-negative LC3B puncta have been reported in trigeminal neurons of HSV1-infected mice but were shown to be cGAMP independent and interferon dependent.9Katzenell S. Leib D.A. Herpes simplex virus and interferon signaling induce novel autophagic clusters in sensory neurons.J Virol. 2016; 90: 4706-4719Crossref PubMed Scopus (36) Google Scholar In contrast, we find this phenomenon to be TBK1 dependent and IFN-β independent because TRIF−/− fibroblasts, shown to have undetectable IRF3 phosphorylation and interferons after HSV1 infection (see Figs E3, A, and E4 in this article's Online Repository at www.jacionline.org),1Sancho-Shimizu V. Pérez de Diego R. Lorenzo L. Halwani R. Alangari A. Israelsson E. et al.Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency.J Clin Invest. 2011; 121: 4889-4902Crossref PubMed Scopus (178) Google Scholar were able to induce this phenotype. Furthermore, interferon treatment was able to induce autophagy in TBK1+/− fibroblasts, ruling out the role of interferon in inducing cytoplasmic LC3B puncta in HSV1 infection (see Fig E5 in this article's Online Repository at www.jacionline.org). TBK1+/− fibroblasts also failed to reduce STING levels after HSV1 infection in contrast to control and TRIF−/− fibroblasts, suggesting autophagy induction by HSV1 may be STING dependent (see Fig E3, B). We confirmed similar HSV1-induced autophagy phenotypes in primary fibroblasts from which these SV40-immortalized cell lines were derived (see Fig E6 in this article's Online Repository at www.jacionline.org). These results show that the 2 types of autophagy differ in localization (cytoplasmic vs perinuclear) and temporal response to HSV1 infection, implying that they have different functions. We decided to focus on the TBK1-dependent early cytoplasmic phenotype as the later perinuclear LC3B, likely nuclear envelope-derived autophagy, was induced in all cells and has been reported to be a generalized stress response to viral late protein production.8Radtke K. English L. Rondeau C. Leib D. Lippe R. Desjardins M. Inhibition of the host translation shutoff response by herpes simplex virus 1 triggers nuclear envelope-derived autophagy.J Virol. 2013; 87: 3990-3997Crossref PubMed Scopus (28) Google Scholar We next sought to understand how the different triggers of autophagy affect HSV1 infection. After pretreatment with poly(I:C), HSV1 replication was significantly reduced in control fibroblasts (nontreated: 5.8 × 105, poly[I:C] treated: 5.5 × 104, P = .006), which can be attributed to production of IFN-β (Fig 2, C and D). Consistently, with a low dose of HSV1, no viral plaque was observed in control fibroblasts, which exhibited cytoplasmic puncta in response to the poly(I:C) treatment (Fig 2, C-E and G). Interestingly, poly(I:C)-induced LC3B puncta in TBK1+/− fibroblasts was detectable after HSV1 infection. However, this pre-enhanced autophagy and IFN-β production in TBK1+/− fibroblasts did not improve cell viability or viral replication in contrast to control fibroblasts (Fig 2, C-G). TRIF−/− fibroblasts did not induce autophagy or interferons after poly(I:C) treatment1Sancho-Shimizu V. Pérez de Diego R. Lorenzo L. Halwani R. Alangari A. Israelsson E. et al.Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency.J Clin Invest. 2011; 121: 4889-4902Crossref PubMed Scopus (178) Google Scholar and hence were not protected against HSV1 infection (Fig 2, D). Notably, however, cytoplasmic LC3B puncta was present following HSV1 infection of TRIF−/− fibroblasts, confirming that formation of cytoplasmic LC3B puncta is interferon independent (Fig 2, C, D, and G, and see Fig E3, A, and E4). Rapamycin pretreatment led to induction of autophagy in control, TRIF−/−, and TBK1+/− fibroblasts, as expected (Fig 2, C and G). In control and TRIF−/− fibroblasts, upregulating autophagy with rapamycin before HSV1 infection resulted in the same proportion of cytoplasmic LC3B puncta after infection (nontreated infected vs rapamycin infected; Fig 2, C and G). In contrast, rapamycin-pretreated TBK1+/− fibroblasts showed a 4-fold (nontreated infected: 8.7% vs rapamycin infected: 35.5%) increase of cytoplasmic LC3B puncta in antigen-negative fibroblasts after HSV1 infection (Fig 2, C and G). Rapamycin pretreatment did not affect viral replication in all cells; however, it significantly improved cell viability of TBK1+/− cells (nontreated: 70.0% vs rapamycin treated: 76.7%, P = .04) and TRIF−/− cells (nontreated: 55.0% vs rapamycin treated: 66.8%, P = .003; Fig 2, E-G). Taken together, this shows that rapamycin-induced autophagy selectively increased the level of cytoplasmic LC3B, which confers a cytoprotective effect by increasing cell viability in TBK1+/− fibroblasts. This protective effect of rapamycin could not be attributed to IFN-β because rapamycin did not induce IFN-β (Fig 2, D). In conclusion, we show that in addition to its antiviral role in interferon production through TLR3 and STING,1Sancho-Shimizu V. Pérez de Diego R. Lorenzo L. Halwani R. Alangari A. Israelsson E. et al.Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency.J Clin Invest. 2011; 121: 4889-4902Crossref PubMed Scopus (178) Google Scholar, 2Herman M. Ciancanelli M. Ou Y.-H. Lorenzo L. Klaudel-Dreszler M. Pauwels E. et al.Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood.J Exp Med. 2012; 209: 1567-1582Crossref PubMed Scopus (138) Google Scholar, 6Burdette D.L. Monroe K.M. Sotelo-Troha K. Iwig J.S. Eckert B. Hyodo M. et al.STING is a direct innate immune sensor of cyclic di-GMP.Nature. 2011; 478: 515-518Crossref PubMed Scopus (1015) Google Scholar TBK1 induces autophagy following HSV1 infection. We demonstrate that TBK1-induced autophagy occurs early during HSV1 infection in antigen-negative fibroblasts, can be mediated by c-di-GMP or HSV1 dsDNA, and is TLR3 and interferon independent. TBK1+/− fibroblasts derived from a patient with HSE harboring a dominant negative mutation had a selective impairment of autophagy induction early in infection represented by the lack of cytoplasmic LC3B puncta formation. We believe that host or virus-induced factors, possibly acting as danger signals, can trigger autophagy in antigen-negative fibroblasts, promoting cell survival without influencing viral replication. This study highlights a possibly cytoprotective role for TBK1 in HSV1-induced autophagy, which might serve to control inflammation and has potential implications for patients with HSE. Human SV40-immortalized dermal fibroblasts from healthy control subjects, TBK1+/− (p.G159A) patients, and TRIF−/− patientsE1Sancho-Shimizu V. Pérez de Diego R. Lorenzo L. Halwani R. Alangari A. Israelsson E. et al.Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency.J Clin Invest. 2011; 121: 4889-4902Crossref PubMed Scopus (236) Google Scholar, E2Herman M. Ciancanelli M. Ou Y.-H. Lorenzo L. Klaudel-Dreszler M. Pauwels E. et al.Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood.J Exp Med. 2012; 209: 1567-1582Crossref PubMed Scopus (203) Google Scholar and Vero (African green monkey kidney) cells were maintained in a 5% CO2 incubator at 37°C in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS. Human fibroblasts were infected with HSV1–green fluorescent protein (KOS strain with green fluorescent protein–tagged capsid protein VP26) or HSV1 (strain 17AR+) at various MOIs and time points for immunoblot and immunofluorescence experiments. After 1 hour of infection in DMEM supplemented with 2% FBS, the virus was removed, and fresh media added with 1% human serum. Viral titers were determined by infecting a confluent monolayer of Vero cells in a 12- or 96-well plate and performing a plaque assay or calculating the 50% end point (TCID50/mL).E3Reed L.J. Muench H. A simple method of estimating fifty per cent endpoints.Am J Hyg. 1938; 27: 493-497Google Scholar Fibroblast cells were stimulated with 25 μg/mL of poly(I:C; GE Healthcare, Chicago, Ill), 10 nmol/L rapamycin (Calbiochem, San Diego, Calif), or 1 × 105 IU/mL of IFN-α-2A (PBL Assay Science, Piscataway, NJ) for 24 hours; or treated with 1 μmol/L of the TBK1 inhibitor BX795 (Sigma, St Louis, Mo)E4Clark K. Plater L. Peggie M. Cohen P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.J Biol Chem. 2009; 284: 14136-14146Crossref PubMed Scopus (280) Google Scholar for 16 hours; or transfected with 8 μg/mL c-di-GMP (InvivoGen, San Diego, Calif) or c-di-GMP control (InvivoGen) for 2 hours; or 2 μg/mL HSV1 dsDNA (60mer sequence: 5′-TAAGACACGATGCGATAAAATCTGTTTGTAAAATTTATTAAGGGTACAAATTGCCCTAGC-3′; Integrated DNA Technology, Coralville, Iowa) for 3 hours. c-di-GMP, c-di-GMP control, and HSV1 dsDNA were delivered using Lipofectamine 2000. For pretreatment experiments, fibroblasts were incubated with either rapamycin or poly(I:C) for 16 hours before infection with HSV1. Cells were plated in a flat-bottom 96-well plate in triplicate at a density of 0.18 × 106 cells/mL in 10% FBS-supplemented DMEM. Fibroblasts were pretreated for 16 hours before infection with HSV1 (MOI 1) for 24 hours. Fibroblast viability was measured using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) Kit (Promega, Madison, Wis) and performed according to the manufacturer's instructions. Cell viability was determined by normalizing to noninfected cells of each cell line. Cells were seeded in 10% FBS-supplemented DMEM at 0.05 × 106 cells/well in a flat-bottomed 24-well plate and incubated for 18 hours in a 37°C humidified incubator. Medium was then replaced with fresh 2% FBS-supplemented DMEM, and cells were transfected with 15 nmol/L scramble (siNegative; Ambion, Thermo Fisher Scientific, Waltham, Mass) or 3 pooled TBK1-specific (siTBK1) siRNAs (siRNA ID nos. 134003, 134002, 899; Ambion) at 80% confluency using Lipofectamine RNAiMAX (Life Technologies, Grand Island, NY) and incubated for a further 48 hours in a 37°C incubator. Cells were grown to 50% confluency on 13-mm-diameter coverslips and fixed with 100% methanol on ice before washing with 1 × PBS. Blocking was done in 10% FBS-PBS, and cells permeabilized with 0.1% Triton X-100 in PBS. Staining was performed in a moist chamber using the following antibodies: LC3B (1:500 dilution; Abcam, Cambridge, United Kingdom), cleaved LC3A (1:100 dilution; StraTech, Eden Prairie, Minn), HSV1-immediate early protein ICP4 (clone 10F1; 1:500 dilution; Virusys, Taneytown, Md), and Alexa Fluor 488– and Alexa Fluor 594–conjugated secondary antibodies (1:750 and 1:1000 dilutions, respectively; Life Technologies). Images were taken with a 63 × oil-immersion lens on a widefield fluorescence microscope (Axio Observer; Zeiss, Oberkochen, Germany). The number of LC3B puncta-positive cells was quantified on a minimum number of 100 cells per experiment. Whole-cell lysates of fibroblasts infected with HSV1 for 48 hours were harvested in 1 × Laemmli buffer supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany), 10% β-mercaptoethanol, and 1:1000 benzonase nuclease (Sigma). Lysates were denatured and electrophoresed on 12% tris-glycine or 10% bis-tris gels (Bio-Rad Laboratories, Hercules, Calif). Proteins were transferred onto polyvinylidene difluoride membrane (Invitrogen, Carlsbad, Calif), probed with primary and secondary horseradish peroxidase–conjugated antibodies (1:1,000 and 1:10,000 dilution, respectively), and subsequently detected using enzyme chemiluminescent reagents (GE Healthcare). For LC3B immunoblots, bafilomycin A1 (Sigma) was added to culture medium for 6 hours before lysis for immunoblotting to block LC3BII recycling to LC3BI. The following antibodies were used for immunoblotting: LC3B (Cell Signaling Technology), p62 (MBL International, Woburn, Mass), TBK1 (Cell Signaling Technology), IRF3 (D83B9; Cell Signaling Technology), phosphorylated IRF3 (S396; Cell Signaling Technology), STING (D2P2F; Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–horseradish peroxidase (Santa Cruz Biotechnology, Dallas, Tex). GAPDH was used as a loading control. IFN-β secretion in recovered supernatants of cells infected with HSV1 or pretreated with rapamycin or poly(I:C) for 24 hours was measured with the VeriKine-HS human IFN-β serum ELISA kit (assay range, 1.2–150 pg/mL; PBL Assay Science), according to the manufacturer's instructions. Immunofluorescence images were analyzed with Icy software. Densitometric analyses of immunoblots were carried out with ImageJ software (National Institutes of Health, Bethesda, Md). Statistical significance was assessed by using 2-way ANOVA or the Student t test with Prism 7 software (GraphPad Software, La Jolla, Calif). Numbers of fluorescent cells or LC3B puncta–positive cells were quantified with ImageJ software. All experiments were performed at least 3 times.Fig E2TRIF−/− fibroblasts showed delayed cytoplasmic LC3B puncta formation. A, Fibroblasts grown on coverslips were infected with HSV1 (MOI 10) for indicated lengths of time before being fixed and stained for LC3B (green), HSV1 ICP4 (red), or both. 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate cytoplasmic LC3B, whereas yellow arrows indicate perinuclear LC3B. B, The percentage of cells positive for cytoplasmic LC3B puncta was counted on at least 100 cells. Images are representative of 3 independent experiments (n = 3). Data are represented as means ± SEMs and were analyzed by using 2-way ANOVA. ***P < .001 and ****P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Immunoblots of TBK1, IRF3, phosphorylated IRF3, and STING during HSV1 infection. Control, TRIF−/−, and TBK1+/− fibroblasts were infected with HSV1 (MOI 1) for the indicated lengths of time. Whole-cell lysates were electrophoresed and probed for endogenous TBK1, IRF3, and phosphorylated IRF3 (p-IRF3; A) and STING (B) proteins. GAPDH was used as a loading control. Relative level of STING to GAPDH was measured using densitometry (arbitrary units).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4TRIF−/− and TBK1+/− fibroblasts showed impaired IFN-β production after HSV1 infection. Control, TRIF−/−, and TBK1+/− fibroblasts were infected with HSV1 at indicated MOIs for 24 hours before collecting supernatants and measuring IFN-β levels by ELISA. Data are represented as means ± SEMs and were analyzed by using 2-way ANOVA (n = 3). *P < .05 and ****P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Interferon-induced autophagy in fibroblasts. A, Control, TRIF−/−, and TBK1+/− fibroblasts were stimulated with 1 × 105 IU/mL IFN-α-2A for 24 hours before being fixed and stained for LC3B (green). 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate LC3B puncta. Images are representative of 3 independent experiments (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Primary fibroblasts showed similar HSV1-induced autophagy phenotypes to SV40-transformed fibroblasts. Primary fibroblasts grown on coverslips were infected with HSV1 (MOI 10) for 3 or 8 hours before being fixed and stained for LC3B (green), HSV1 ICP4 (red), or both. 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate cytoplasmic LC3B, whereas yellow arrows indicate perinuclear LC3B. Images are representative of 3 independent experiments (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

DNA sensors including cGAS, STING and IFI16 are key components of the innate immune response to infection. However, the precise mechanisms of action, in particular the relative importance of direct suppression of replication versus paracrine signalling of an antiviral state to susceptible cells remains unclear. We examined the kinetics of herpes simplex virus infection and spread in a relevant cell type, human keratinocytes, lacking one or other of these DNA sensors using time-lapse microscopy. We also examine transcriptional induction of interferon from the native locus, at single cell and single molecule level using highly sensitive RNA FISH. Our results reveal distinct aspects of the roles of these factors and reveal outcomes not appreciated by other methods. Cells lacking either of these factors showed increased susceptibility to initial infection (prior to any downstream paracrine signalling) but with quite different outcomes. Lack of cGAS resulted in increased cellular migration and cell density at the infection focus. On the other hand, cells lacking STING showed lower cell density and significantly increased cytopathic effect likely curtailing virus yield. Initial results demonstrate that we can analyse interferon transcription at single cell level with exquisite sensitivity down to a few transcripts per cell and reveal profound spatial heterogeneity in responses to induction by PAMP ligands. Altogether, our results reveal new insight into the spatial landscape of the initiation and spread of HSV and key cellular responses which likely integrate pathways including innate immunity, apoptosis and cell migration.

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.

We used the bioorthogonal protein precursor, homopropargylglycine (HPG) and chemical ligation to fluorescent capture agents, to define spatiotemporal regulation of global translation during herpes simplex virus (HSV) cell-to-cell spread at single cell resolution. Translational activity was spatially stratified during advancing infection, with distal uninfected cells showing normal levels of translation, surrounding zones at the earliest stages of infection with profound global shutoff. These cells further surround previously infected cells with restored translation close to levels in uninfected cells, reflecting a very early biphasic switch in translational control. While this process was dependent on the virion host shutoff (vhs) function, in certain cell types we also observed temporally altered efficiency of shutoff whereby during early transmission, naïve cells initially exhibited resistance to shutoff but as infection advanced, naïve target cells succumbed to more extensive translational suppression. This may reflect spatiotemporal variation in the balance of oscillating suppression-recovery phases. Our results also strongly indicate that a single particle of HSV-2, can promote pronounced global shutoff. We also demonstrate that the vhs interacting factor, eIF4H, an RNA helicase accessory factor, switches from cytoplasmic to nuclear localisation precisely correlating with the initial shutdown of translation. However translational recovery occurs despite sustained eIF4H nuclear accumulation, indicating a qualitative change in the translational apparatus before and after suppression. Modelling simulations of high multiplicity infection reveal limitations in assessing translational activity due to sampling frequency in population studies and how analysis at the single cell level overcomes such limitations. The work reveals new insight and a revised model of translational manipulation during advancing infection which has important implications both mechanistically and with regards to the physiological role of translational control during virus propagation. The work also demonstrates the potential of bioorthogonal chemistry for single cell analysis of cellular metabolic processes during advancing infections in other virus systems.

CREB-H, an ER-anchored transcription factor, plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes, including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases, releasing the N-terminal product, which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif termed the P-motif, located in the N-terminus between serines 73 and 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation, promoting ER retention, while mutation of target serines and drug inhibition of GSK-3 activity coordinately induce both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations, which regulate stability by targeting the protein to the SCF Fbw1a E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilization of the active form in the nucleus.

We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC binding to solid supports that allowed coupling to external capture agents and demonstrated that the vast majority of individual virions contained bioorthogonally-tagged genomes. Using HSVEdC in vivo we reveal novel aspects of the kinetics, localisation, mechanistic entry requirements and morphological transitions of infecting genomes. Uncoating and nuclear import was observed within 30 min, with genomes in a defined compaction state (ca. 3-fold volume increase from capsids). Free cytosolic uncoated genomes were infrequent (7-10% of the total uncoated genomes), likely a consequence of subpopulations of cells receiving high particle numbers. Uncoated nuclear genomes underwent temporal transitions in condensation state and while ICP4 efficiently associated with condensed foci of initial infecting genomes, this relationship switched away from residual longer lived condensed foci to increasingly decondensed genomes as infection progressed. Inhibition of transcription had no effect on nuclear entry but in the absence of transcription, genomes persisted as tightly condensed foci. Ongoing transcription, in the absence of protein synthesis, revealed a distinct spatial clustering of genomes, which we have termed genome congregation, not seen with non-transcribing genomes. Genomes expanded to more decondensed forms in the absence of DNA replication indicating additional transitional steps. During full progression of infection, genomes decondensed further, with a diffuse low intensity signal dissipated within replication compartments, but frequently with tight foci remaining peripherally, representing unreplicated genomes or condensed parental strands of replicated DNA. Uncoating and nuclear entry was independent of proteasome function and resistant to inhibitors of nuclear export. Together with additional data our results reveal new insight into the spatiotemporal dynamics of HSV genome uncoating, transport and organisation.