Finger Domain Mutation Affects Enzyme Activity, DNA Replication Efficiency, and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

Abstract

The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G ⅐ C sites during the polymerization reaction. The implications of the findings are discussed.Herpes simplex virus (HSV) DNA polymerase consists of the catalytic subunit of the polymerase (Pol) and the processivity factor UL42. The Pol subunit contains three well-defined activities: polymerization (replication), exonuclease proofreading (editing), and UL42 binding (5, 6, 28). The UL42 binding activity is mediated by amino acid residues located at the C terminus (5, 6). Although the UL42 binding residues are unique to certain alphaherpesvirus DNA polymerases, the sequences comprising the polymerase and exonuclease domains are conserved among the B family (or the ␣-like) polymerases (2-4). The exonuclease domain of the HSV type 1 (HSV-1) Pol contains conserved exonuclease I (Exo I), II, and III motifs, whereas the polymerase domain contains seven conserved regions (I to VII); conserved region IV overlaps with the Exo II motif. The Exo III motif is located within the ␦ region C, which is highly conserved among the B family polymerases (Fig. 1A). These conserved regions are located within the palm, the thumb, and the finger subdomains, which comprise the structural components of the polymerase domain. The crystal structure of the HSV-1 Pol subunit revealed three grooves that form the putative polymerase, exonuclease, and DNA binding sites. The putative exonuclease site is defined as a groove formed between the exonuclease domain...

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

“…The 3=-5= exonuclease and lyase active sites have been mapped within the interior of the 3=-5= exonuclease domain (17,22) and a 63-kDa C-terminal fragment (3), respectively, and are separate from the sites of our engineered mutations within the extreme N-terminal 51 residues at the surface of the enzyme. Additionally, a previous study had reported that a mutant lacking detectable 3=-5= exonuclease activity exhibited a 50-fold decrease in viral yield with only a 3-fold decrease in viral DNA production (40). The defect in viral yield observed in the previous study largely reflected an alteration in replication fidelity rather than a defect in viral DNA synthesis, as seen with our Pol mutants (40).…”

“…Additionally, a previous study had reported that a mutant lacking detectable 3=-5= exonuclease activity exhibited a 50-fold decrease in viral yield with only a 3-fold decrease in viral DNA production (40). The defect in viral yield observed in the previous study largely reflected an alteration in replication fidelity rather than a defect in viral DNA synthesis, as seen with our Pol mutants (40). Regardless, purification and assay of Pol⌬N52 and PolA 6 proteins relative to WT Pol confirmed that the mutations did not induce any global effects on protein folding, as the mutant proteins exhibited robust 5=-3= polymerase activity.…”

Mechanisms of viral mutation

“…A HSV-1 polymerase mutant containing Y577H/D581A substitutions was exonuclease-deficient and exhibited a mutator phenotype. However, this variant rapidly evolved a compensatory substitution (L774F) that restored DNA replication fidelity in this genetic background [ 49 , 50 ]. Since RNA virus polymerases typically lack this activity, no such mutators can be produced, except for coronaviruses [ 51 ].…”

Antimutator variants of DNA polymerases

“…Conserved polymerase and exonuclease motifs are indicated by colored frames: blue, Exo motifs; green, Pol motifs (Wang et al , 1989); brown, motifs A, B, C (Delarue et al , 1990). Amino-acid substitutions of interest are placed above or below the alignment at the relevant position, highlighted according to the following scheme: aqua, T4 Pol antimutators (Reha-Krantz, 1995a; Reha-Krantz and Wong, 1996; Reha-Krantz, 2010); magenta, HSV1 Pol antimutators (Hall et al , 1984; Gibbs et al , 1988; Hwang et al , 2004; Tian et al , 2009); yellow, yeast pol3-01 ; no highlight, antimutators isolated as suppressors of the pol3-01 mutator phenotype; green, antimutators isolated as suppressors of the pol3-D407A mutator phenotype; orange, antimutators isolated as suppressors of the pol3-F406A mutator phenotype (three substitutions in the same mutant); blue, antimutator isolated as a suppressor of the pol3-D463A mutator phenotype; gray, previously reported antimutators in Pol δ ( pol3-t (D643N), G447S, Y708A, and V758M) (Tran et al , 1999a; Hadjimarcou et al , 2001; Pavlov et al , 2004; Li et al , 2005). The antimutators affecting pol3-01 , pol3-D407A , pol3-F406A , and pol3-D463A are from Herr et al (Herr et al , 2011).…”