G1/S Cell Cycle Induction by Epstein-Barr Virus BORF2 Is Mediated by P53 and APOBEC3B.

Herpesvirus lytic infection causes cells to arrest at the G1/S phase of the cell cycle by poorly defined mechanisms. In a prior study using fluorescent ubiquitination-based cell cycle indicator (FUCCI) cells that express fluorescently tagged proteins marking different stages of the cell cycle, we showed that the Epstein-Barr virus (EBV) protein BORF2 induces the accumulation of G1/S cells, and that BORF2 affects p53 levels without affecting the p53 target protein p21. We also found that BORF2 specifically interacted with APOBEC3B (A3B) and forms perinuclear bodies with A3B that prevent A3B from mutating replicating EBV genomes. We now show that BORF2 also interacts with p53 and that A3B interferes with the BORF2-p53 interaction, although A3B and p53 engage distinct surfaces on BORF2. Cell cycle analysis showed that G1/S induction by BORF2 is abrogated when either p53 or A3B is silenced or when an A3B-binding mutant of BORF2 is used. Furthermore, silencing A3B in EBV lytic infection increased cell proliferation, supporting a role for A3B in G1/S arrest. These data suggest that the p53 induced by BORF2 is inactive when it binds BORF2, but is released and induces G1/S arrest when A3B is present and sequesters BORF2 in perinuclear bodies. Interestingly, this mechanism is conserved in the BORF2 homologue in HSV-1, which also re-localizes A3B, induces and binds p53, and induces G1/S dependent on A3B and p53. In summary, we have identified a new mechanism by which G1/S arrest can be induced in herpesvirus lytic infection. IMPORTANCE In lytic infection, herpesviruses cause cells to arrest at the G1/S phase of the cell cycle in order to provide an optimal environment for viral replication; however, the mechanisms involved are not well understood. We have shown that the Epstein-Barr virus BORF2 protein and its homologue in herpes simplex virus 1 both induce G1/S, and do this by similar mechanisms which involve binding p53 and APOBEC3B and induction of p53. Our study identifies a new mechanism by which G1/S arrest can be induced in herpesvirus lytic infection and a new role of APOBEC3B in herpesvirus lytic infection.

Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma.

Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR] domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.

Triple-mutated oncolytic herpes virus for treating both fast- and slow-growing tumors.

Oncolytic virus therapy has emerged as a promising treatment option against cancer. To date, oncolytic viruses have been developed for malignant tumors, but the need for this new therapeutic modality also exists for benign and slow-growing tumors. G47∆ is an oncolytic herpes simplex virus type 1 (HSV-1) with an enhanced replication capability highly selective to tumor cells due to genetically engineered, triple mutations in the γ34.5, ICP6 and α47 genes. To create a powerful, but safe oncolytic HSV-1 that replicates efficiently in tumors regardless of growth speed, we used a bacterial artificial chromosome system that allows a desired promoter to regulate the expression of the ICP6 gene in the G47∆ backbone. Restoration of the ICP6 function in a tumor-specific manner using the hTERT promoter led to a highly capable oncolytic HSV-1. T-hTERT was more efficacious in the slow-growing OS-RC-2 and DU145 tumors than the control viruses, while retaining a high efficacy in the fast-growing U87MG tumors. The safety features are also retained, as T-hTERT proved safe when inoculated into the brain of HSV-1 sensitive A/J mice. This new technology should facilitate the use of oncolytic HSV-1 for all tumors irrespective of growth speed.

Viral Induced Protein Aggregation: A Mechanism of Immune Evasion.

Various intrinsic and extrinsic factors can interfere with the process of protein folding, resulting in protein aggregates. Usually, cells prevent the formation of aggregates or degrade them to prevent the cytotoxic effects they may cause. However, during viral infection, the formation of aggregates may serve as a cellular defense mechanism. On the other hand, some viruses are able to exploit the process of aggregate formation and removal to promote their replication or evade the immune response. This review article summarizes the process of cellular protein aggregation and gives examples of how different viruses exploit it. Particular emphasis is placed on the ribonucleotide reductases of herpesviruses and how their additional non-canonical functions in viral immune evasion are closely linked to protein aggregation.

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

Attenuated Herpes Simplex Virus 1 (HSV-1) Expressing a Mutant Form of ICP6 Stimulates a Strong Immune Response That Protects Mice against HSV-1-Induced Corneal Disease.

We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39, resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0-, ICP8-, and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8- virus, but not the ICP0- virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases.IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0-) virus and a replication-incompetent (ICP8-) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development.

Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense.

The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domain-containing adapter inducing IFN-ß, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6-RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1-infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism.

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins.

The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries.

Species-Specific Inhibition of Necroptosis by HCMV UL36.

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.

ICP6 Prevents RIP1 Activation to Hinder Necroptosis Signaling.

Necroptosis is a type of programmed necrosis which depends on the activation of receptor-interacting protein kinase 3 (RIP3). Herpes simplex virus type 1 (HSV-1) is known to block necroptosis by the viral protein ICP6 in human cells, but its specific inhibitory mechanism is not fully understood. Here we reported that ICP6 could promote rather than suppress the formation of necrosome, the necroptosis signaling complex containing RIP3 and upstream regulator receptor-interacting protein kinase 1 (RIP1), but blocked RIP3 activation. Moreover, ICP6 could reduce the necroptosis-specific auto-phosphorylation of RIP1 regardless of the presence of RIP3. These results indicate that ICP6 block necroptosis through preventing RIP1 activation dependent signal transduction in necrosome.