Exonuclease-Deficient Polymerase Mutant of Herpes Simplex Virus Type 1 Induces Altered Spectra of Mutations

Hwang, Ying T.; Hwang, Charles B. C.

doi:10.1128/jvi.77.5.2946-2955.2003

Cited by 17 publications

(10 citation statements)

References 25 publications

(33 reference statements)

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“…These mutant Pols exhibited defective or undetectable exonuclease activity, yet retained substantial polymerase activity. Furthermore, recombinant viruses containing the mutated Pol displayed only modestly defective replication in Vero cells and exhibited substantially increased mutation frequencies in the viral tk (23) and supF (25,27) genes accompanying altered spectra of mutations (23,29). Further characterization of these Pol mutants demonstrated that the Y7 exonuclease-deficient Pol mutant could generate heterogeneous viruses containing mutations in genes coding for DNA replicative proteins, including Pol and its accessory protein (23) UL42 (26).…”

Section: Resultsmentioning

confidence: 99%

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A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

Hwang

Zuccola

Lü

et al. 2004

J Virol

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Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.DNA polymerase (Pol) is the pivotal enzyme involved in DNA replication and plays a central role in controlling the accuracy of DNA replication. Amino acid sequence alignment of various Pols reveals that all Pols contain conserved residues distributed within several regions of the Pol domain. Previous studies demonstrated that these conserved residues play an important role in the polymerization reaction (reviewed in reference 38 and references therein). These conserved residues interact with incoming deoxyribonucleotides (deoxynucleoside triphosphates [dNTPs]) and the primer-template DNA. Therefore, mutations within these residues could result in altered polymerase activity and/or replication fidelity.Herpes simplex virus (HSV) has proven to be a good model for genetic, molecular biological, and biochemical studies of Pol, since it can be manipulated to contain desired mutations for further characterization regardless of whether the mutated Pol is lethal for viral replication (reviewed in reference 11 and references therein). Examination of the HSV-1 Pol also reveals seven conserved regions within the carboxyl-terminal half of the Pol, which has been defined as the polymerase domain (Fig. 1). Indeed, mutations within most of these conserved regions confer altered sensitivities to certain nucleoside and pyrophosphate analog antiviral drugs, indicating that they are involved in the interactions with dNTPs and DNA (11).Since only a few region VI mutants of HSV have been described (4, 5, 34), and the effects of these mutations on replication fidelity have not been characterized, we constructed recombinant HSV-1 viruses containing a region VI mutation (L774F) for characterizing the effects of this mutatio...

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Section: Resultsmentioning

confidence: 99%

“…The mutagenesis assay and data analyses were performed as described previously (23,25). Briefly, 2 ϫ 10 4 Vero cells were infected with recombinant virus at less than 200 PFU for 72 h. Total DNA, including that prepared from virions present in the supernatants, was extracted, purified, and…”

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A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

Hwang

Zuccola

Lü

et al. 2004

J Virol

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“…That study (13) reported that selectivity of correct over incorrect dNTP was ϳ300, whereas bacteriophage T4 and T7 DNA polymerases discriminate among dNTPs by a factor of Ͼ1000 and Ͼ100,000, respectively (14 -16). Previous studies suggested that the exo activity of HSV-1 pol contributes substantially to the fidelity of HSV replication in vivo, because mutation of the conserved exo site III of the HSV-1 pol catalytic subunit was associated with a strong mutator phenotype (10,17,18). Moreover, such pol mutants are genetically unstable and revert or recombine with high frequency (10), suggesting strong in vivo selection against an HSV-1 pol lacking exo activity.…”

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Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

Song

Chaudhuri

Knopf

et al. 2004

Journal of Biological Chemistry

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Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3 -to 5 -exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K m for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wildtype pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol⅐UL42 complex was much less efficient than by the mutant pol⅐UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol⅐UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.Herpes simplex virus type 1 (HSV-1) 1 is the best characterized member of the large family of Herpesviridae pathogenic to humans, which also includes Epstein-Barr virus, varicella-zoster virus, human cytomegalovirus, and Kaposi sarcoma-associated herpes virus (reviewed in Ref. 1). Viruses in this family encode most of the proteins essential for and directly involved in DNA replication (2-4), including a well conserved DNA polymerase catalytic subunit (pol), which is a member of the polymerase B family (5, 6). HSV-1 pol possesses 5Ј-to 3Ј-polymerizing and 3Ј-to 5Ј-exonuclease (exo) activities (7,8), the latter of which is involved in the removal of incorrectly incorporated deoxyribonucleoside triphosphates (9 -12). The importance of this proofreading activity for maintaining fidelity of DNA replication was suggested by studies from our laboratory that demonstrated the relatively poor ability of HSV-1 pol to discriminate between the correct and incorrect nucleotide for incorporation in single turnover experiments (13). That study (13) reported that selectivity of correct o...

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“…Accordingly, HSV-1 Pol exhibits 5=-3= polymerase and 3=-5= exonuclease activities that are characteristic functions of the replicative Pol B family (20,26,35). Polymerase activity is essential for the production of infectious progeny virus (1,13), while the 3=-5= exonuclease activity is not required for viral replication but is important for replication fidelity during viral DNA synthesis (16,17). The extreme C terminus of HSV-1 Pol is crucial for an interaction with processivity factor UL42 that is necessary for long-chain DNA synthesis and indispensable for viral replication (11).…”

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

Terrell

Coen

2012

J Virol

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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.

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Exonuclease-Deficient Polymerase Mutant of Herpes Simplex Virus Type 1 Induces Altered Spectra of Mutations

Cited by 17 publications

References 25 publications

A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

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

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Exonuclease-Deficient Polymerase Mutant of Herpes Simplex Virus Type 1 Induces Altered Spectra of Mutations

Cited by 17 publications

References 25 publications

A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

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

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