Periodic Reporting for period 1 - HSV1_Single_Molecule (Single molecule imaging of herpes simplex virus type 1 DNA replication)
Berichtszeitraum: 2022-09-01 bis 2024-08-31
Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen with a seroprevalence of about 65% globally. The symptoms caused by an HSV-1 infection are most commonly mucosal lesions around the mouth (cold sores) and in rare cases inflammation of the brain (meningitis) or eyes (keratitis). Other medically relevant human pathogens can be found within the same virus family including varicella zoster virus, Epstein-Barr virus, cytomegalovirus and Kaposi sarcoma herpesvirus. Despite extensive research into herpesvirus biology, we still lack a complete understanding of the mechanism of herpesvirus genome replication. Genome replication is a fundamental process of any organism or virus. A profound understanding of the intricate mechanisms of viral genome replication will allow us to develop targeted antiviral drugs in the future.
We here revisited long-standing questions of the HSV-1 genome replication mechanism using cutting-edge single molecule imaging techniques. This approach enables the detection of transient structures and heterogeneous behaviour of individual molecules, leading to crucial insights into this fundamental process, which could not be gained using more conventional assays. The overall objective of this project was to gain a deeper understanding of herpesvirus replication mechanism by addressing unanswered questions in the field using latest imaging technology.
One of the objectives of this project was to assess the DNA structures, which are formed upon viral replication on the single-molecule level. We therefore first established an assay using TIRF microscopy to enable this. Another objective was to study the properties of the HSV-1 origin binding protein using novel imaging techniques.
We here revisited long-standing questions of the HSV-1 genome replication mechanism using cutting-edge single molecule imaging techniques. This approach enables the detection of transient structures and heterogeneous behaviour of individual molecules, leading to crucial insights into this fundamental process, which could not be gained using more conventional assays. The overall objective of this project was to gain a deeper understanding of herpesvirus replication mechanism by addressing unanswered questions in the field using latest imaging technology.
One of the objectives of this project was to assess the DNA structures, which are formed upon viral replication on the single-molecule level. We therefore first established an assay using TIRF microscopy to enable this. Another objective was to study the properties of the HSV-1 origin binding protein using novel imaging techniques.
During this project we first established a range of different single-molecule imaging assays to address open questions in herpes simplex virus type 1 (HSV-1) genome replication. These include techniques such as single-molecule FRET, TIRF microscopy, c-trap and cryoEM. We used these techniques to address long-standing questions in the field.
We visualised the formation of different structures during HSV-1-mediated DNA replication thereby revealing a long anticipated mechanism of replication in real-time. Our assay showed the HSV-1-mediated formation of a long continuous DNA molecules consisting of multiple copies of the template DNA (concatemer) in a coordinated manner. We further addressed the mechanism of initiation of replication by the origin binding protein and discovered a previously unknown property of the protein. This result supports an alternative hypothesis for replication initiation within the field. The results of this project have been communicated at national and international scientific meetings and will be published in a peer-reviewed journal.
We visualised the formation of different structures during HSV-1-mediated DNA replication thereby revealing a long anticipated mechanism of replication in real-time. Our assay showed the HSV-1-mediated formation of a long continuous DNA molecules consisting of multiple copies of the template DNA (concatemer) in a coordinated manner. We further addressed the mechanism of initiation of replication by the origin binding protein and discovered a previously unknown property of the protein. This result supports an alternative hypothesis for replication initiation within the field. The results of this project have been communicated at national and international scientific meetings and will be published in a peer-reviewed journal.
We successfully introduced single-molecule imaging techniques to study herpesvirus genome replication. This has, to our knowledge, not been done before and is anticipated to yield groundbreaking results in the future. Using these techniques we were able to answer some of the long-standing questions in the field and gain novel insights into the mechanism of replication. Understanding this important phase during the viral life cycle is crucial to develop novel antiviral drugs. We further showed that single molecule imaging techniques are powerful tools to study viral genome replication and thereby open up new avenues to study replication of other viruses.