Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

ABSTRACTHerpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination ofde novoassembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensivein vitropassaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion toin vivopatient infection studies.IMPORTANCEHerpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.

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RNA Polymerase II Promoter-Proximal Pausing and Release to Elongation Are Key Steps Regulating Herpes Simplex Virus 1 Transcription

Journal of Virology ◽

10.1128/jvi.02035-19 ◽

2019 ◽

Vol 94 (5) ◽

Cited By ~ 3

Author(s):

Claire H. Birkenheuer ◽

Joel D. Baines

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Rna Polymerase Ii ◽

Viral Genome ◽

Viral Dna ◽

Herpes Simplex Virus 1 ◽

Pol Ii ◽

Late Genes ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (Pol II). Expression of viral immediate early (α) genes is followed sequentially by early (β), late (γ1), and true late (γ2) genes. We used precision nuclear run-on with deep sequencing to map and to quantify Pol II on the HSV-1(F) genome with single-nucleotide resolution. Approximately 30% of total Pol II relocated to viral genomes within 3 h postinfection (hpi), when it occupied genes of all temporal classes. At that time, Pol II on α genes accumulated most heavily at promoter-proximal pause (PPP) sites located ∼60 nucleotides downstream of the transcriptional start site, while β genes bore Pol II more evenly across gene bodies. At 6 hpi, Pol II increased on γ1 and γ2 genes while Pol II pausing remained prominent on α genes. At that time, average cytoplasmic mRNA expression from α and β genes decreased, relative to levels at 3 hpi, while γ1 relative expression increased slightly and γ2 expression increased more substantially. Cycloheximide treatment during the first 3 h reduced the amount of Pol II associated with the viral genome and confined most of the remaining Pol II to α gene PPP sites. Inhibition of both cyclin-dependent kinase 9 activity and viral DNA replication reduced Pol II on the viral genome and restricted much of the remaining Pol II to PPP sites. IMPORTANCE These data suggest that viral transcription is regulated not only by Pol II recruitment to viral genes but also by control of elongation into viral gene bodies. We provide a detailed map of Pol II occupancy on the HSV-1 genome that clarifies features of the viral transcriptome, including the first identification of Pol II PPP sites. The data indicate that Pol II is recruited to late genes early in infection. Comparing α and β gene occupancy at PPP sites and gene bodies suggests that Pol II is released more efficiently into the bodies of β genes than α genes at 3 hpi and that repression of α gene expression late in infection is mediated by prolonged promoter-proximal pausing. In addition, DNA replication is required to maintain full Pol II occupancy on viral DNA and to promote elongation on late genes later in infection.

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Herpes Simplex Virus Nuclear Nonvirion Antigens Detected by Anticomplement Immunofluorescence

Tumori Journal ◽

10.1177/030089167606200604 ◽

1976 ◽

Vol 62 (6) ◽

pp. 609-614 ◽

Cited By ~ 1

Author(s):

Giulio Tarro ◽

Giovan Giacomo Giordano ◽

Armando Tripodi ◽

Romolo Cerra ◽

Mario Di Gioia ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Genome ◽

Squamous Cell Carcinomas ◽

Nuclear Antigen ◽

New Approach ◽

Human Sera ◽

Simplex Virus ◽

And Control

The finding of a nuclear antigen by anticomplement immunofluorescence in cells treated with cytosine-arabinoside after infection of Herpes Simplex Virus (HSV), opens a new approach to the problem of the role of this virus in certain human cancers. Complement-fixing tests of HSV markers with cancer and control human sera as well as with hyperimmune guinea pig antisera are discussed, suggesting another parameter for studies of squamous cell carcinomas. The finding of HSV antigens in selected tumors as the expression of repressed viral genome proves a continuing release of virus specific message and supports the important role of the virus in the development of the tumor.

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Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26

Journal of Virology ◽

10.1128/jvi.02528-05 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8211-8224 ◽

Cited By ~ 93

Author(s):

Katinka Döhner ◽

Kerstin Radtke ◽

Simone Schmidt ◽

Beate Sodeik

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Capsid Protein ◽

Herpes Simplex Virus Type ◽

Viral Genome ◽

Wild Type ◽

Nuclear Targeting ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.

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Herpes Simplex Virus 1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia To Cause Recurrent Ocular Disease

Journal of Virology ◽

10.1128/jvi.00468-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8383-8391 ◽

Cited By ~ 20

Author(s):

Sungseok Lee ◽

Angela M. Ives ◽

Andrea S. Bertke

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Recurrent Disease ◽

Clinical Disease ◽

Ocular Infection ◽

Sensory Ganglia ◽

Autonomic Ganglia ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.IMPORTANCEHerpes simplex virus 1 (HSV-1) and HSV-2 establish latent infections, from which the viruses reactivate to cause recurrent disease throughout the life of the host. However, the viruses exhibit different manifestations and frequencies of recurrent disease. HSV-1 and HSV-2 establish latency in both sensory and autonomic ganglia. Autonomic ganglia are more responsive than sensory ganglia to stimuli associated with recurrent disease in humans, such as stress and hormone fluctuations, suggesting that autonomic ganglia may play an important role in recurrent disease. We show that HSV-1 can reactivate from autonomic ganglia, independently from sensory ganglia, to cause recurrent ocular disease. We found no evidence that HSV-2 could reactivate from autonomic ganglia independently from sensory ganglia after ocular infection, but HSV-2 did replicate in both ganglia simultaneously to cause persistent disease. Thus, viral replication and reactivation in autonomic ganglia contribute to different clinical disease manifestations of HSV-1 and HSV-2 after ocular infection.

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Dynamic Response of IFI16 and Promyelocytic Leukemia Nuclear Body Components to Herpes Simplex Virus 1 Infection

Journal of Virology ◽

10.1128/jvi.02249-15 ◽

2015 ◽

Vol 90 (1) ◽

pp. 167-179 ◽

Cited By ~ 31

Author(s):

Roger D. Everett

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Genome ◽

Viral Gene Expression ◽

Nuclear Body ◽

Viral Gene ◽

Intrinsic Resistance ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTIntrinsic immunity is an aspect of antiviral defense that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus, the balance between cellular repressors and virus-counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus 1 (HSV-1) is conferred by components of promyelocytic leukemia nuclear bodies (PML NBs), which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins, including IFI16, which has been implicated in sensing pathogen DNA and initiating signaling pathways that lead to an interferon response, also respond to viral genomes in this manner. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrated that this response is extremely rapid, occurring within the first hour after addition of the virus, and that human Daxx (hDaxx) and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral-genome-induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures, thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0.IMPORTANCEHerpesviruses have intimate interactions with their hosts, with infection leading either to the productive lytic cycle or to a quiescent infection in which viral gene expression is suppressed while the viral genome is maintained in the host cell nucleus. Whether a cell becomes lytically or quiescently infected can be determined through the competing activities of cellular repressors and viral activators, some of which counteract cell-mediated repression. Therefore, the events that occur within the earliest stages of infection can be of crucial importance. This paper describes the extremely rapid response to herpes simplex virus 1 infection of cellular protein IFI16, a sensor of pathogen DNA, and also of the PML nuclear body proteins PML and hDaxx, as revealed by live-cell microscopy. The data imply that these proteins can accumulate on or close to the viral genomes in a sequential manner which may lead to their sequestration and repression.

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Molecular Aspects of Herpes Simplex Virus I Latency, Reactivation, and Recurrence

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411980090040901 ◽

1998 ◽

Vol 9 (4) ◽

pp. 541-562 ◽

Cited By ~ 46

Author(s):

C.S. Miller ◽

R.J. Danaher ◽

R.J. Jacob

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Recurrent Disease ◽

Epithelial Tissue ◽

Molecular Events ◽

Current Thinking ◽

Molecular Aspects ◽

Simplex Virus ◽

Relationship Of ◽

Hsv 1

The application of molecular biology in the study of the pathogenesis of herpes simplex virus type 1 (HSV-1) has led to significant advances in our understanding of mechanisms that regulate virus behavior in sensory neurons and epithelial tissue. Such study has provided insight into the relationship of host and viral factors that regulate latency, reactivation, and recurrent disease. This review attempts to distill decades of information involving human, animal, and cell culture studies of HSV-1 with the goal of correlating molecular events with the clinical and laboratory behavior of the virus during latency, reactivation, and recurrent disease. The purpose of such an attempt is to acquaint the clinician/scientist with the current thinking in the field, and to provide key references upon which current opinions rest.

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The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores

Journal of Virology ◽

10.1128/jvi.00641-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 12

Author(s):

Jamie B. Huffman ◽

Gina R. Daniel ◽

Erik Falck-Pedersen ◽

Alexis Huet ◽

Greg A. Smith ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Capsid Protein ◽

Viral Genome ◽

Wild Type Virus ◽

Nuclear Pore ◽

Nuclear Pores ◽

Viral Dna ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.

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Role of the Herpes Simplex Virus CVSC Proteins at the Capsid Portal Vertex

Journal of Virology ◽

10.1128/jvi.01534-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Alexis Huet ◽

Jamie B. Huffman ◽

James F. Conway ◽

Fred L. Homa

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Genome ◽

Virus Assembly ◽

Unit Length ◽

Structural Studies ◽

Herpes Simplex Virus 1 ◽

Critical Step ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit-length HSV genome. Here, biochemical and structural studies focused on the unique portal vertex of wild-type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal proteins pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

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SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

Journal of Virology ◽

10.1128/jvi.00426-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 5939-5952 ◽

Cited By ~ 26

Author(s):

James R. Brown ◽

Kristen L. Conn ◽

Peter Wasson ◽

Matthew Charman ◽

Lily Tong ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Genome ◽

Dependent Manner ◽

Intrinsic Immunity ◽

Herpes Simplex Virus 1 ◽

Sumo Ligase ◽

Nuclear Domains ◽

Simplex Virus ◽

Hsv 1

ABSTRACTAspects of intrinsic antiviral immunity are mediated bypromyelocyticleukemianuclearbody (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is thesmallubiquitin-likemodifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligaseproteininhibitor ofactivatedSTAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in aSUMOinteractionmotif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation.IMPORTANCEAdaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.

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