The Pre-NH ₂ -Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

Abstract

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH 2 -terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH 2 -terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5=-3= polymerase activity. We identified three pre-NH 2 -terminal Pol mutants that exhibited 5=-3= polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants Pol⌬N43 and Pol⌬N52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA 6 , in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the pol⌬N43 mutant displayed replication kinetics similar to those of wild-type virus, while pol⌬N52 and polA 6 mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both pol⌬N52 and polA 6 viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH 2 -terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.

Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

“…Interestingly, the HSV-1 DNApol has an extra domain, the pre-NH 2 -terminal domain, according to the three dimensional structure published by Liu et al (32). This domain is required for efficient viral replication as well as for establishment of latency (as observed experimentally in mice) (33,34). In EBV DNApol, the pre-NH 2 -terminal domain is also important for lytic genome replication (35).…”

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

“…The pre-N terminal domain also contains the FYNPYL motif specific to herpesviridae family polymerases (Appendix Figure A1) [9]. It has recently been shown that this motif is required for efficient replication of viral DNA synthesis in vivo ; a mutant polymerase lacking the FYNPYL motif showed a substantial reduction in viral DNA synthesis [34]. However, the purified FYNPYL deletion mutant showed no reduction in polymerase activity, suggesting that this motif may have a function in the formation of the viral DNA replisome.…”

Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3′-to-5′ Direction and Is Dependent on the 3′-to-5′ Exonuclease Active Site

“…To test whether the RNase H activity of HSV-1 Pol requires an active 3=-to-5= exonuclease catalytic site, we analyzed the in vitro activities of purified WT Pol and the previously characterized exonuclease-deficient single mutant, D368A Pol (17,18). We expressed 6ϫHis-tagged full-length WT Pol and 6ϫHis-tagged D368A Pol separately using baculovirus expression (22) and purified these enzymes for in vitro characterization. Next, we assessed the 5=-to-3= polymerization function of both enzymes by testing the ability of these Pols and control enzymes to extend a hairpin template (S1) with a 5= overhang and a 5= fluorescent label ( Fig.…”

“…Protein expression and purification. 6ϫHis-tagged full-length WT HSV Pol and a previously characterized 3=-to-5= exonuclease-deficient mutant, D368A Pol (17,18), were cloned into the pFastBac HTC vector and expressed in a baculovirus system as previously described (22) and then purified as follows. Cells were harvested at 65 h postinfection and centrifuged at 2,800 ϫ g for 30 min.…”