Characterization of Epstein-Barr Virus Subversion of the Host SMC5/6 Restriction Pathway - Abstract
Epstein-Barr virus (EBV) establishes lifelong infection in 95% of adults worldwide. EBV
causes multiple cancers, particularly in hosts with HIV co-infection. EBV is transmitted between
hosts through saliva, from which it translocates across the oral cavity and tonsillar epithelium to
reach the B-cell compartment. In persons with AIDS, EBV lytic replication causes oral hairy
leukoplakia. Upon B-cell infection, the double-stranded DNA (dsDNA) EBV genome is delivered
to the nucleus. Much remains to be learned about host pathways that sense incoming EBV
genomes, as well as viral genomes in cells undergoing lytic replication, and how EBV interacts
with these restriction pathways. We used RNAseq and multiplexed tandem mass tag (TMT)
mass spectrometry to create temporal proteomic maps of primary human B-cell infection and of
EBV lytic replication. Each highlighted that EBV strongly and specifically downmodulates the
SMC5/6 cohesin complex within 1 day of primary B-cell infection, prior to the expression of most
viral genes, and again upon lytic reactivation. We identified that the EBV major tegument protein
BNRF1 assembles a Cullin-7 based ubiquitin ligase that targets SMC5/6 for proteasomal
degradation. In the absence of BNRF1, SMC5/6 shuts down EBV lytic replication compartments
and blocks EBV lytic DNA synthesis. CRISPR SMC6 knockout rescues replication compartment
formation in Burkitt cells carrying BNRF1 knockout EBV. To our knowledge, SMC5/6 is therefore
the first host factor that can restrict EBV replication compartments. However, little is known
about how SMC5/6 recognizes EBV genomes as foreign. This important question has remained
open across double stranded DNA viral genomes, where focus has instead been on how
viruses evade SMC5/6. Our central hypothesis is that the SMC5/6 cohesin recognizes and
restricts viral non-B-form DNA structures necessary for EBV latency establishment and
lytic DNA replication in a manner modulated by BNRF1. Our Aims are to (1) Identify how
SMC5/6 senses and restricts latent EBV DNA in newly infected cells and to (2) Identify how
SMC5/6 senses and restricts lytic EBV DNA upon reactivation. Collectively, these studies
address the key open question about how SMC5/6 functions in order to discriminate viral from
host DNA, which has remained nearly unstudied despite growing recognition of SMC5/6 roles in
regulation of double stranded viral DNA.