Functional analysis of multiple activities associated with the herpes simplex virus-1 DNA polymerase

Abstract

Of the DNA replication proteins required for herpes simplex virus-1 (HSV-1) viral replication, the catalytic subunit of the DNA polymerase (Pol) has been the most extensively studied, both as a model for Family B polymerases and as a successful antiviral target. Despite decades of work, however, there are still questions regarding the enzyme and its functions that have not been fully resolved, especially related to the RNase H activity, the 3’-5’ exonuclease activity, and a putative RNA-binding motif with no defined activity. We set out to answer some of these questions and further define the activities associated with HSV Pol. There has been a controversy regarding whether an RNase H activity intrinsic to HSV-1 Pol is due to the 3’-5’ exonuclease of Pol or whether it is a separate activity, possibly acting on 5’ RNA termini. We found that the RNase H activity of HSV Pol exclusively degrades RNA:DNA hybrids with 3’ RNA ends, and that this activity is dependent upon the 3’-5’ exonuclease active site. Next, we set out to study a 3’-5’ exonuclease-deficient mutant that had been previously studied in vitro but not characterized in cell culture. We confirmed that this mutant was incapable of replicating in non-complementing cells and determined that this substitution severely affected expression of the protein during infection. Finally, we made substitutions within the putative RNA-binding (RNP) motif within the NH2-terminal domain of Pol. We identified that this mutant was incapable of viral replication and viral DNA synthesis, with no defects in expression, localization, or polymerase activity, suggesting that this motif plays a role in viral DNA synthesis separate from the polymerase activity. Together, these data answered several unresolved questions related to the HSV-1 DNA polymerase and identified the RNP motif as crucial for viral DNA synthesis. Our results argue that the RNP motif’s function during viral DNA synthesis is not a 5’-3’ RNase H. Additionally, we were able to provide evidence that the defects in replication associated with a well-studied 3’-5’ exonuclease mutant were due to expression and not the exonuclease per se.