Sequencing of HSV-1 Exo mutants and WT genomes isolated from different passages in Vero cells

Herpes simplex virus type 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions were extensively studies in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separated. Consequently, exonuclease activity can be reduced or abolished by certain point mutations withing catalytically important regions, however, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, recovery of HSV-1 mutants with a catalytically inactive exonuclease was so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, were believed to be lethal. Here we show that HSV-1 can be recovered in absence of intrinsic Pol exonuclease activity and demonstrate that a lack of proofreading causes rapid accumulation of mostly detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain.