Suicidal Phenotype of Proofreading-Deficient Herpes Simplex Virus 1 Polymerase Mutants

Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely 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. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.