A group led by Louis Flamand, PhD in Canada has developed a culture system that can be used to determine how HHV-6 enters latency by integrating into the chromosome, and which drugs cause it to activate.
Unlike the other herpesviruses that curl up into an episome in the nucleus when they enter latency, HHV-6A integrates into the chromosome as a form of latency. Therefore, the reactivation models developed for other herpesviruses are not applicable to HHV-6 & HHV-7.
Flamand’s group developed two culture systems, one with individual clones and a second using bulk cultures and ddPCR. They found that following infection, 1-22% of the clones tested contained integrated virus, depending on the cell line and viral stock used. The viral copy number per cell ranged from 0.2 to 4.3. The authors assume that cells containing less than one copy is likely due to the fact that the integrated virus is lost in some cells while clones with >1 copy/cell may have had multiple integration events.
Gene expression in Flamand’s culture system was both spontaneous and induced. Immediate early 1 expression was detected in 10-20% of cells. In addition, clones stimulated by compounds such as TSA, hydrocortisone, and TPA showed strong immediate-early, early, and late gene expression, although other genes such as U54 and U94 were not efficiently activated.
Flamand also confirmed that multiple integrations can occur. They found that HHV-6B could integrate into 9% of clones already integrated with HHV-6A, and HHV-6A could integrate into 38% of clones with ciHHV-6B.
The investigators also found that HHV-6 can integrate into most of the cell lines tested, and that using a compound that inhibits DNA replication did not affect HHV-6A/B integration. They also found that certain drugs such as HDAC inhibitors could trigger the expression of many viral genes, without the production of infectious virus.
Among the surprising findings: integration can occur during non-productive infection, and the virus often leaves the tips of the genome behind when it exits the chromosome.
A lab led by Bhupesh Prusty, PhD in Germany previously reported a new culture system for studying HHV-6A latency and reported that HHV-6 can integrate into human chromosomes in the absence of DNA replication (Gulve 2017).
Read the full paper: Gravel 2017.