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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - VGAP (The Viral Genome Associated Proteome)

Teaser

Summary

Herpesviruses have evolved sophisticated ways to subvert the immune system during millions of years of coevolution with their respective hosts. The nine human members of the herpesvirus family include important human pathogens like Herpes-simplex viruses-1 and -2 (HSV-1 and HSV-2), Varicella-Zoster Virus (VZV), Human Cytomegalovirus (HCMV), Human Herpesviruses-6A, -6B and -7 and the tumor viruses Epstein-Barr virus (EBV) and Kaposi\'s sarcoma associated herpesvirus (KSHV). A hallmark of herpesvirus infections is the establishment of lifelong latency, for example about 3.7 billion people alone are infected with HSV-1 worldwide, and even more by HHV-6, -7 and EBV.
The goal of this project was the identification of novel restriction or host factors of herpesviruses that are involved or interfere with initiation of herpesviral gene expression and replication. Therefore a new method called HyCCAPP should be developed in order to discover new factors that associate with the viral genome upon nuclear entry. Although the initially proposed approach was not successful, we used alternative methods, namely CRISPRainbow- and EdC-labelling of viral genomes that allowed us to identify SMCHD1 as a novel restriction factor for herpesviruses that is associated with viral genomes. SMCHD1 is an important human protein, and mutations in SMCHD1 have been shown to be the cause of two genetic diseases, Facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphtalmia syndrome (BAMS). We characterized SMCHD1 in detail as antiviral protein and could even show viral antagonism of SMCHD1-mediated restriction by a protein of KSHV, further corroborating the importance of SMCHD1 in antiviral immunity. We hope that our discovery of a new antiviral restriction factor may help to understand the pathogenicity of herpesviral disease and contribute to the development of new anti-herpesviral drugs in the future.

Work performed

We proposed to use the Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP) method to identify novel proteins associated with herpesviral DNA in the nucleus, the viral genome associated proteome or VGAP. HyCCAPP was developed in a yeast system, however in our infection system we were not able to precipitate enough cellular proteins. We immediately implemented our risk management strategy and therefore we adapted two alternative methods, the recently developed CRISPRainbow technology and EdC-labelling of viral genomes followed by click-chemistry to visualize the chromatin of HSV-1 genomes in cells. We confirmed the functionality of both methods by visualization of viral genomic DNA in confocal microscopy and colocalization with a HSV-1-encoded protein that binds to replicating viral genomes.
For proteomic profiling of infected cells, EdC-labelling of viral genomes worked best and we continued our experiments using this method. One protein that interacted with the viral genome and attracted our attention was Structural maintenance of chromosomes (SMC) flexible hinge domain-containing 1 (SMCHD1). SMCHD1 is associated with the viral genome within the first 4h post infection. SMCHD1 is a chromatin protein involved in epigenetic silencing and two very different developmental diseases has been attributed to SMCHD1: FSHD and BAMS. SMCHD1 has been implicated in various epigenetic processes and in particular X-chromosome inactivation. In addition, SMCHD1 also plays a role in the DNA-damage response, in particular in the decision whether a DNA double strand break is repaired by homologous recombination repair (HR-repair) or non-homologous end-joining (NHEJ). SMCHD1 blocks HR-repair and promotes NHEJ. We hypothesize that the viruses need to block the cellular DDR immediately after infection to prevent repair of incoming linear genomes by NHEJ.
We could confirm the association of SMCHD1 with the HSV-1 genome 2h post infection and could show that SMCHD1 levels decrease over the course of HSV-1 infection However upon infection with KSHV, no decrease of SMCHD1 protein could be observed, therefore we hypothesized that KSHV might encode for a viral protein that antagonizes SMCHD1 function. In order to investigate the role of SMCHD1 during viral infection we next generated cell lines with depletion of SMCHD1 using the CRISPR/CAS9 system and sgRNAs specific for SMCHD1. Upon knockout of SMCHD1 we observed an increase in HSV-1 infected cells compared to wt-cells. However we could not detect any effect of SMCHD1-knockout on the replication of KSHV. We therefore hypothesized that KSHV might encode for a protein that antagonizes SMCHD1 function and could identify ORF75 of KSHV as viral antagonist of SMCHD1. We found that expression of ORF75 led to a relocalization of SMCHD1. ORF75 is a member of a well-known viral protein family with homology to cellular enzymes. The so called viral-FGARATs have been shown to counteract the antiviral function of innate immune compartments in the nucleus of cells. To our own surprise, we found that ORF75 not only inhibits SMCHD1 function but is also a broad inhibitor of the DNA-damage response in general.
In summary, our data show that SMCHD1 is a general antiviral factor against infection with herpesviruses. We hypothesize that blocking SMCHD1 function by ORF75 immediately after infection creates a nuclear environment favouring homologous recombination. This is beneficial for herpesviruses because it promotes genome replication and prevents loss of viral genetic information. Taken together, our data support an important role for SMCHD1 in the epigenetic regulation of incoming viral genomes and establish SMCHD1 as a novel intrinsic restriction factor for herpesviruses.

Final results

\"Parts of our results were already published in a peer-reviewed open access publication in the journal \"\"Nature Microbiology\"\", which is ranked the #2 original research journal with an impact factor of 14.3 in the category microbiology according to Clarivate Analytics Journal Citation Report. The main part of our project will be published soon, and we aim again to publish our results in a prestigious journal with open access.
In addition, the press office of the University Hospital Erlangen released two press announcements about the project that led to several articles and interviews in German and international newspapers and magazines about the research. These also drew the attention of many patients suffering from herpesviral infection. Although we cannot offer treatments and medical advice for patients, this underlines the need for new antiherpesviral treatments and the impact of related research.
Our project discovered a new herpesviral restriction factor. Although SMCHD1 is the cause of two genetic diseases (FSHD and BAMS), its role in the control of herpesviral infection is a novel finding. It would very interesting to investigate whether SMCHD1-mediated restriction is specific for herpesviruses or a general mechanism for DNA-viruses, or even RNA viruses that replicate inside the nucleus like influenza. In addition, the interaction of the viral genome with SMCHD1 provides a possible target for therapeutic intervention as antivirals against herpesviral diseases are urgently needed in the clinics. We hope that our findings will stimulate more research on the topic and will be for the benefit of patients in the future.

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