Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin.

Fourteen mutants known or likely to contain mutations in the herpes simplex virus DNA polymerase gene were examined for their sensitivity to aphidicolin in plaque reduction assays. Eleven of these exhibited some degree of hypersensitivity to the drug; altered aphidicolin-sensitivity correlated with altered sensitivity to the pyrophosphate analog, phosphonoacetic acid. The DNA polymerase specified by one of these mutants, PAAr5, required roughly seven-fold less aphidicolin to inhibit its activity by 50% than did polymerase specified by its parental strain. Mutations responsible for the aphidicolin-hypersensitivity phenotype of PAAr5 were mapped to an 0.8 kbp region in the herpes simplex virus DNA polymerase locus. These data taken together indicate that 1) mutations in the herpes simplex virus DNA polymerase gene can confer altered sensitivity to aphidicolin, 2) that the HSV polymerase is sensitive to aphidicolin in vivo, and 3) that amino acid alterations which affect aphidicolin binding may affect the pyrophosphate exchange-release site as well, suggesting that aphidicolin binds in close proximity to this site.     

A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin.

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.     

Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene.

Three herpes simplex virus mutants that contain drug resistance mutations in the DNA polymerase gene exhibited no significant reduction in replication in the ears of mice compared with the wild type after inoculation at that site but were attenuated for pathogenicity after intracerebral inoculation. Cataracts were common sequelae in mice that survived mutant infections.     

Unusual regulation of expression of the herpes simplex virus DNA polymerase gene.

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.     

Aphidicolin resistance in herpes simplex virus type 1 appears to alter substrate specificity in the DNA polymerase.

We describe novel mutants of herpes simplex virus which are resistant to aphidicolin. Their mutant phenotypes suggest that they encode DNA polymerases with altered substrate recognition. This conclusion is based on their abnormal sensitivity to polymerase inhibitors and to the abnormal mutation rates exhibited by two of the mutants.     

Mutations of the woodchuck hepatitis virus polymerase gene that confer resistance to lamivudine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil.

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.     

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.     

Mutations in the DNA polymerase and thymidine kinase genes of herpes simplex virus clinical isolates resistant to antiherpetic drugs

Primary structures of DNA polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates di ffering in sensitivity to several antiherpetic drugs were determined and compared to those of two laboratory HSV-1 strains one of which (L₂) was sensitive and the other (L2/R) was resistant to acyclovir. The phylogenetic sequence analysis showed that the ul30 and ul23 sequences of clinical isolates were close to those of L2, and that ul30 conserved regions differed between HSV-1 isolates and L₂ only in point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA polymerase and thymidine kinase functional domains were identified as substitutions associated with strain resistance to ACV and other antiherpetic drugs.     

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.     

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.     

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.     

Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity.

We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.     

Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site

We describe the mapping and sequencing of mutations within the DNA polymerase gene of herpes simplex virus type 1 which confer resistance to aphidicolin, a DNA polymerase inhibitor. The mutations occur near two regions which are highly conserved among DNA polymerases related to the herpes simplex enzyme. They also occur near other herpes simplex mutations which affect the interactions between the polymerase and deoxyribonucleoside triphosphate substrates. Consequently, we argue in favor of the idea that the aphidicolin binding site overlaps the substrate binding site and that the near-by conserved regions are functionally required for substrate binding. Our mutants also exhibit abnormal sensitivity to another DNA polymerase inhibitor, phosphonoacetic acid. This drug is thought to bind as an analogue of pyrophosphate. A second-site mutation which suppresses the hypersensitivity of one mutant to phosphonoacetic acid (but not its aphidicolin resistance) is described. This second mutation may represent a new class of mutations, which specifically affects pyrophosphate, but not substrate, binding.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

The herpes simplex virus DNA polymerase: Analysis of the functional domains

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597–961, comprising the homologous sequence regions IV–IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3′-to-5′ exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 ± 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3′-to-5′ exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072–1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.