Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments.

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.

Direct DNA sequencing of PCR products.

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam-Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by "asymmetric PCR," where one primer is present in vast excess over the other. This single-stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.

Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo.

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAAr5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker gene and the HSV oris sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAAr5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G. C-to-T. A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases.

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

Mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene can confer altered drug sensitivities.

Two herpes simplex virus mutants containing mutated residues within the conserved Exo III motif of the polymerase gene were previously shown to be defective in 3'-5' exonuclease activity and exhibited extremely high mutation frequencies. In this study, we have shown that these mutants also exhibited higher resistance to phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs tested, including acyclovir and gangciclovir, compared to wild-type virus. Marker transfer experiments and sequencing analyses demonstrated that these altered phenotypes were the result of mutations within the Exo III motif. The data indicate that, aside from leading to exonuclease deficiency, mutations in the Exo III motif may also affect interaction of nucleoside triphosphates with the catalytic sites of polymerase activity.

Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity.

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3'-5' exonuclease activities, contains sequence motifs that are homologous to those important for 3'-5' exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3'-5' exonuclease contributes substantially to the fidelity of viral DNA replication.

Absorption of irrigating fluid during transcervical resection of endometrium--a report of two cases.

It has been recognized for many years that the use of hypotonic solution for the irrigation of the bladder cavity during transurethral resection of the prostate (TURP) may result in hyponatremia and water intoxication due to rapid and excessive absorption of the solution from the exposed prostatic bed, the clinical manifestation of which is termed "TURP syndrome". A similar condition termed "female TURP syndrome" following hysteroscopic transcervical endometrial resection (TCR) has been reported. Since the frequency of TCR continues to increase the increased rate of "TCR syndrome" would come in its wake. Here, we present two cases who developed severe hyperglycemia and hyponatremia while underwent TCR with 10% dextrose in water as the irrigation fluid and the same time emphasize the potential risk of this complication.

Unexpected left endobronchial intubation in a case of Turner's syndrome.

Patients with Turner's syndrome present a multiplicity of cardiovascular and airway abnormalities. We describe a case of Turner's syndrome with pericardial effusion who experienced an inadvertent endobronchial intubation due to displacement of the endotracheal tube immediately after the pericardial drainage. A sudden increase of airway pressure and decrease of oxygen saturation in the presence of breathing sound audible via the chest piece placed on the left hemithorax first misled us to call our attention to sputum impaction. No sputum was obtainable from airway suction. Inadvertent bronchial intubation was highly suspected at the post-anesthesia room when a decrease of right side breathing sound was noted together with a progressive fall of oxygen saturation. Emergent chest x-ray confirmed the diagnosis. It was thought that the mishap took place early in the act of or following the pericardial drainage. Our case serves as evidence once again to emphasize the possibility of endobronchial intubation due to displacement of endotracheal tube during anesthesia and the importance of monitoring to guard against inadvertent bronchial intubation, particularly in patients with associated problems such as Turner's syndrome with huge pericardial effusion.

An altered spectrum of herpes simplex virus mutations mediated by an antimutator DNA polymerase.

The mutation spectrum attributable to an antimutator DNA polymerase in a eukaryotic cell was examined. Drug-resistant thymidine kinase (tk) mutants derived from both a wild-type (wt) strain, KOS and an antimutator DNA polymerase (pol) mutant, PAAr5, of herpes simplex virus (HSV), were isolated, and the mutated tk genes were characterized at the sequence level. While both transition and frameshift mutations were found in the mutated tk genes derived from the wt KOS Pol, all the PAAr5-mediated mutants analyzed were frameshift mutations. These results imply that the wt HSV Pol is less faithful than the antimutator enzyme, at least in part, because of its propensity to mediate transition mutations.

Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis.

The 1235 residue herpes simplex virus DNA polymerase is a prototype alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The C-terminal fragments bound the viral polymerase processivity factor, UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertinent to other DNA polymerases.

A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.

Clinical resistance to antiviral drugs requires that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants of herpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and translation of mutant tk genes, including constructs in which reporter epitopes could be expressed only if frameshifting occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading frame cloning failed to detect alterations in tk transcripts that could account for the synthesis of full-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frameshift during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.

Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia.

From marker rescue, sequencing, transcript, and latency analyses of the thymidine kinase-negative herpes simplex virus mutant dlsactk and studies using the thymidine kinase inhibitor Ro 31-5140, we infer that the virus-encoded thymidine kinase is required in murine trigeminal ganglia for acute replication and lytic gene expression, for increasing the numbers of cells expressing latency-associated transcripts, and for reactivation from latent infection.

Functions and proteins of herpes simplex virus type-1 that are involved in raising the mutation frequency of infected cells.

When cells are infected by herpes simplex virus type-1 (HSV-1) the mutation frequency is increased. To find which functions of the virus are responsible for this, a variety of viral strains and one fragment of viral DNA were tested in a mutagenesis assay. Mutagenesis was dependent on the binding of the virus to the cell surface and disassembly of the virus particle, but expression of virus genes was not necessary. Since this implied that mutagenesis was a result of the exposure of the interior of the cell to an internal structural component of the virus, the role of two likely components was examined. The host-shutoff function of the virus was not required for mutagenesis. However, a fragment of DNA from within the minimum transforming region of HSV-1 that encodes a possible virion protein was mutagenic when expressed from a eukaryotic expression vector. The encoded product of this DNA fragment is therefore a candidate for a transforming protein of HSV-1, and is the only protein currently suggested to be responsible for that function.

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.

Analysis of complex mutations induced in cells by herpes simplex virus type-1.

The shuttle vector plasmid pZ189 was used as a target for mutagenesis in COS-1 cells. Complex mutations were analyzed in 5 plasmids that were recovered from noninfected cells and 15 plasmids that were recovered from cells that were infected with herpes simplex virus type-1 (HSV-1). Complex mutations in noninfected cells consisted of duplications and rearrangements of plasmid DNA, while those from cells that were infected with HSV-1 included 8 that were enlarged due to insertion of other DNA sequences. Plasmids that contained inserted DNA showed an increase in size of from 118 bp up to around 4500 bp. Maps were constructed based on restriction enzyme digestion, and some or all of each plasmid was examined by DNA sequencing. The inserted DNA was not derived from HSV-1 in any case, since it did not hybridize to DNA from HSV-1 and showed no sequence similarities to the virus. Instead, inserted DNA was found to hybridize to HindIII-digested cellular DNA as a single or double band in 5 plasmids and contained multiple repeat sequences such as alpha satellite, Alu or Kpn repeats in 4 plasmids. In four enlarged plasmids the identity of the inserted sequences could not be determined. The junctions between the shuttle vector and the inserted DNA did not show features of transposable elements and no homology was detected between inserted sequences and the sequence at the insertion site. No preferred site for recombination was detected. Although no similarities were found among the inserted sequences, it is possible that the cellular sequences represent cellular targets for virus-mediated rearrangement. It appears that HSV-1 stimulates nonhomologous recombination between DNA sequences in virus-infected cells.

Keratin expression in cultures of adult human epidermal cells.

Keratins are complex fibrous proteins characteristic of epithelial cells. We have developed a procedure that allows us to culture and passage adult human dermal keratinocytes in the absence of mesenchymal substrates. Electron microscopic examination of stratifying cultures showed the presence of numerous filament bundles, desmosomes and electron dense granules. The expression of different classes of keratin was examined by immunofluorescence, SDS-PAGE and immunoblots using monoclonal antibodies. The analysis of water-insoluble proteins revealed the presence of keratins of molecular weights 40 Kda, 50-52 Kda, 56 Kda and 65-67 Kda. Our results indicate that the terminal differentiation of keratinocytes may not require dermal factors.

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.

DNA sequence of mutations induced in cells by herpes simplex virus type-1.

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced in cells by herpes simplex virus type-1 (HSV-1). A significant increase in mutation frequency was detected as early as 2 hr after infection, and reached a peak of two- to sevenfold over background at 4 hr after infection. Several differences were detected between spontaneous mutants and those induced by HSV-1 when they were analyzed by gel electrophoresis and DNA sequencing. Point mutations accounted for 63% of spontaneous mutants but for only 44% of HSV-1-induced mutants (P less than 0.05). In each case the predominant type of point mutation was the G:C to A:T transition, which comprised 51% of point mutations induced by HSV-1, and 32% of spontaneous point mutations. Deletions of DNA were seen in HSV-1-induced mutants at a frequency of 44%, compared with only 29% in spontaneous mutants. HSV-1-induced deletions were less than half the length of spontaneous deletions, and 3 contained short filler sequences. An increase in size was seen in 13% of HSV-1-induced mutants and was due either to duplication of plasmid DNA, or, in 8 instances, to insertion of sequences derived from cellular DNA. Among spontaneous mutants, only 8% were increased in size and none of them had inserted cellular DNA. The proportion of complex mutants increased as infection by the virus progressed and they accounted for 79% of mutants at 24 hr after infection. The observed mutations have implications for understanding the "hit and run" mechanism of malignant transformation of cells by HSV-1.

Shedding of herpes simplex virus type 1 into saliva.

The shedding of herpes simplex virus Type 1 (HSV-1) into saliva was studied in 110 healthy individuals, 55 patients who were undergoing oral surgery, and 21 immunocompromised patients in an asymptomatic Japanese population. Specimens were collected regularly for up to 2 months from each individual. Virus was recovered on at least one occasion from 4.5% of the healthy individuals, 20% of the patients undergoing oral surgery and 38% of the immunocompromised patients. In those individuals that shed virus, the duration of shedding varied from a mean of 1.2 days in the healthy individuals to 5.8 days in patients undergoing oral surgery, and was 3 days in the immunocompromised patients. The majority of oral surgery patients shed HSV-1 into saliva within 7 days after surgery and immunocompromised patients shed virus almost continuously. The role of surgery in triggering the reactivation of latent HSV-1, and the differences in rates of viral shedding between American and Japanese are discussed.

Detection of herpes simplex virus proteins in cultured cells by monoclonal antibodies and the avidin-biotin-immunoperoxidase complex method.

The distribution of 5 proteins of herpes simplex virus Type 1 was observed in cells that had been infected for various periods. The cells were stained with monoclonal antibodies to ICP4, ICP5, ICP6, ICP8, and gB, using the avidin-biotin-peroxidase complex (ABC) method. Each protein had a characteristic pattern of time of appearance and translocation by which it could be distinguished from the others. The sensitivity of the ABC technique, its ease of use, and the permanence of the preparations make this method well suited for the study of viral proteins.