Systematic Enhancement of Human Herpesvirus 6A Reconstitution: Bridging Technical Refinement and Host Determinants of Virus Replication

Human herpesvirus 6A (HHV-6A) is a betaherpesvirus that presents substantial challenges for research due to its strong propensity for latency and slow replication. This thesis addresses the critical bottleneck of efficient recombinant HHV-6A reconstitution from bacterial artificial chromosome (BAC) constructs, a process historically hampered by low transfection efficiencies and prolonged propagation duration. We demonstrated that pretreatment with dimethyl sulfoxide (DMSO) and exonuclease V (ExoV), coupled with refined post-nucleofection cell recovery, increased transfection efficiencies up to 30%. We identified coordinated stimulation strategies that markedly enhanced virus replication. Notably, combining the hypoxia-mimetic IOX2 and hydrocortisone (HC) drastically reduced reconstitution time to two weeks, providing the first evidence that the hypoxic response plays a role in HHV-6A replication. Furthermore, we elucidated the suppressive role of interferon (IFN) signaling in virus reconstitution, highlighting the potential of JAK1/JAK2 inhibition via ruxolitinib (RUX) to improve viral yields. By systematically optimizing nucleofection and stimulation conditions, we developed a robust and reproducible methodology that significantly enhances virus reconstitution. Our refined method, validated across multiple experimental conditions, facilitates the generation of recombinant HHV-6A mutants, including a novel reporter virus for detailed studies of virus replication kinetics and gene functions. Collectively, these advances provide a powerful platform for mechanistic studies of HHV-6A biology and lay the groundwork for future investigations into herpesvirus latency, reactivation and antiviral strategies

Author dc:creator

• Reich, Jana

• human herpesvirus 6
• viral replication
• reconstitution
• stimuli
• enhancers
• laboratory methods

Licence dc:rights.uri

• https://www.fu-berlin.de/sites/ub/publizieren/refubium/rechtliches/index.html

Language dc:language

eng

Identifier URI

http://dx.doi.org/10.17169/refubium-52124

OAI record oai:identifier

https://refubium.fu-berlin.de/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arefubium.fu-berlin.de%3Afub188%2F52394