A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.

We have shown that a drug-resistant mutant from a clinical isolate of herpes simplex virus contains a single point mutation in the DNA polymerase gene that confers resistance to both acyclovir and foscarnet. The mutated amino acid is located within a distinct conserved region shared among alpha-like DNA polymerases which we designate region VII. We infer that these conserved sequences are directly or indirectly involved in the recognition and binding of nucleotide and PPi substrates.     

In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme.

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.     

Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

Exonuclease-deficient polymerase mutant of herpes simplex virus type 1 induces altered spectra of mutations

The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.6%, consistent with the tk mutation frequencies reported previously (Y. T. Hwang, B.-Y. Liu, D. M. Coen, and C. B. C. Hwang, J. Virol. 71:7791-7798, 1997). The increased mutation frequencies were 10- to 500-fold higher than those observed for wild-type Pol recombinants. The increased mutation frequencies also were significantly higher than those of supF mutant replicated by exonuclease-deficient Pols in the plasmid-borne assay. Furthermore, characterization of supF mutants demonstrated that recombinants with a defective exonuclease induced types and distributions of supF mutations different from those induced by wild-type Pol recombinants. The types of supF mutations induced by exonuclease-deficient recombinants differed between the plasmid- and genome-based assays. The spectra of supF mutations also differed between the two assays. In addition, exonuclease-defective viruses also induced different spectra of supF and tk mutations. Therefore, both the assay methods and the target genes used for mutagenesis studies can affect the repication fidelity of herpes simplex virus type 1 Pol with defective exonuclease activity.     

Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.     

The enhanced DNA replication fidelity of a mutant herpes simplex virus type 1 DNA polymerase is mediated by an improved nucleotide selectivity and reduced mismatch extension ability

We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.     

Acyclovir-resistant mutants of herpes simplex virus type 1 express altered DNA polymerase or reduced acyclovir phosphorylating activities.

The biochemical properties of four acyclovir-resistant mutants are described. Two of these mutants, PAAr5 and BWr, specified nucleotidyl transferase (DNA polymerase) activities which were less sensitive to inhibition by acyclovir triphosphate than their wild-type counterparts. Another mutant, IUdRr, exhibited reduced ability to phosphorylate acyclovir. The fourth mutant, ACGr4, both induced an altered DNA polymerase and failed to phosphorylate appreciable amounts of acyclovir. BWr, a new acyclovir-resistant mutant derived from the Patton strain of herpes simplex virus type 1, induced a DNA polymerase resistant to inhibition by acyclovir triphosphate, but, unlike the polymerases induced by PAAr5 and ACGr4, still sensitive to phosphonoacetic acid. Resistance of BWr to acyclovir mapped close to the PAAr locus and was separable from mutations in the herpes simplex virus thymidine kinase gene by recombination analysis.     

Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome

An assay has been developed and used to locate an origin of DNA replication on the herpes simplex virus type 1 (HSV-1) genome. Baby hamster kidney cells were transfected with circular plasmid molecules containing cloned copies of HSV-1 DNA fragments, and helper functions were provided by superinfection with wild-type HSV-1. The presence of an HSV-1 origin of replication within a plasmid enabled amplification of the vector DNA sequences, which was detected by the incorporation of [32P]orthophosphate. By screening various HSV-1 DNA fragments it was possible to identify a 995-bp fragment that maps entirely within the reiterated sequences flanking the short unique region of the viral genome and contains all the cis-acting signals necessary to function as an origin of viral DNA replication. The products of plasmid replication were shown to be high mol. wt. DNA molecules consisting of tandem duplications of the complete plasmid, suggesting that replication was occurring by a rolling-circle mechanism.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Contribution of the 3'- to 5'-exonuclease activity of herpes simplex virus type 1 DNA polymerase to the fidelity of DNA synthesis

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 wild-type 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 pol.UL42 complex was much less efficient than by the mutant pol.UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol.UL42 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.     

Crystal structure of the herpes simplex virus 1 DNA polymerase

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.     

The herpes simplex virus type 1 DNA polymerase processivity factor increases fidelity without altering pre-steady-state rate constants for polymerization or excision

Pre-steady-state and steady-state kinetics of nucleotide incorporation and excision were used to assess potential mechanisms by which the fidelity of the herpes simplex virus type 1 DNA polymerase catalytic subunit (Pol) is enhanced by its processivity factor, UL42. UL42 had no effect on the pre-steady-state rate constant for correct nucleotide incorporation (150 s(-1)) nor on the primary rate-limiting conformational step. However, the equilibrium dissociation constant for the enzyme in a stable complex with primer-template was 44 nm for Pol and 7.0 nm for Pol/UL42. The catalytic subunit and holoenzyme both selected against incorrect nucleotide incorporation predominantly at the level of nucleotide affinity, although UL42 slowed by 4-fold the maximum rate of incorporation of incorrect, compared with correct, nucleotide. Pol, with or without UL42, cleaved matched termini at a slower rate than mismatched ones, but UL42 did not significantly alter the pre-steady-state rate constant for mismatch excision ( approximately 16 s(-1)). The steady-state rate constant for nucleotide addition was 0.09 s(-1) and 0.03 s(-1) for Pol and Pol/UL42, respectively, and enzyme dissociation was the rate-limiting step. The longer half-life for DNA complexes with Pol/UL42 (23 s) compared with that with Pol (8 s) affords a greater probability for excision when a misincorporation event does occur, accounting predominantly for the failure of Pol/UL42 to accumulate mismatched product at moderate nucleotide concentrations.     

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.     

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.     

On the mutation rate of herpes simplex virus type 1

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.