The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Specific herpes simplex virus-induced incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into deoxyribonucleic acid.

5-Iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) is a novel thymidine analog which inhibits herpes simplex virus, type 1 (HS-1 virus) replication in the absence of detectable host toxicity. When murine, simian, or human cells in culture are treated with [125I]AIdUrd for up to 24 hours essentially none of the nucleoside becomes cell-associated. In contrast, upon HS-1 virus infection significant radiolabel is detected in both nucleotide pools and in DNA. The major acid-soluble metabolite has been shown by enzymic and chromatographic analysis to be the 5'-triphosphate of AIdUrd. DNA from HS-1 virus-infected Vero cells labeled with [14C]thymidine, 5-[125I]iodo-2'-deoxyuridine (IdUrd), or [125I]AIdUrd was isolated by buoyant density centrifugation and subjected to digestion by pancreatic DNase I, spleen DNase II, micrococcal nuclease, spleen, and venom phosphodiesterases. Analysis of the digestion products clearly indicate that AIdUrd is incorporated internally into the DNA structure. DNA containing AIdUrd therefore contains phosphoramidate (P-N) bonds, known to be extremely acid-labile. The selective HS-1 virus-induced phosphorylation of AIdUrd and its subsequent incorporation into DNA may account for the unique biological activity of the AIdUrd nucleoside.     

The relationship between incorporation of E-5-(2-Bromovinyl)-2'-deoxyuridine into herpes simplex virus type 1 DNA with virus infectivity and DNA integrity

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) produced a dose-dependent shift in the density of herpes simplex virus type 1 (HSV-1) DNA at concentrations which yielded potent inhibition of virus replication in cultured Vero cells. Although the density of cellular DNA was not altered by these concentrations of BrvdUrd, incorporation of this analogue into cellular DNA of HSV-1-infected cells has been previously observed in this laboratory. The degree of inhibition correlated with the amount of BrvdUrd substituted for thymidine in HSV-1 DNA. BrvdUrd-substituted DNA was more labile as determined by a dose-dependent increase in single strand breaks when examined by centrifugation in alkaline sucrose gradients. Thus, the potent antiviral action of BrvdUrd observed in cell culture correlates not only with its incorporation into HSV-1 DNA but also with an altered stability of this DNA.     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.     

CTL are inactivated by herpes simplex virus-infected cells expressing a viral protein kinase

Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of U(S)3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.     

5-Fluoro-2'-deoxyuridine incorporation in L1210 DNA

We have employed cesium sulfate density gradient centrifugation to separate RNA and DNA of L1210 cells labeled with [3H]fluorodeoxyuridine. We have analyzed nucleotide and nucleoside digests of purified DNA from the [3H]fluorodeoxyuridine-labeled cells and demonstrate by reverse phase and anion exchange high pressure liquid chromatography the presence of tritium radioactivity co-migrating with fluorodeoxyuridine 5'-monophosphate or fluorodeoxyuridine. These observations demonstrate the internucleotide incorporation of fluorodeoxyuridine in DNA and suggest a new mechanism of action for this cytotoxic and mutagenic agent.     

Tetrahydrouridine specifically facilitates deoxycytidine incorporation into herpes simplex virus DNA.

As reported by Jamieson and Subak-Sharpe (J. Gen. Virol. 31:303-313, 1976), exogenous deoxycytidine is very poorly incorporated into herpes simplex virus DNA. Here it is shown that this incorporation was dramatically increased in the presence of tetrahydrouridine (THU), a specific inhibitor of cytidine-deoxycytidine deaminase. Thus, the exclusion of deoxycytidine from herpes simplex virus DNA probably results from massive degradation by the deaminase, which is consistent with the observation that in the absence of THU, most of the nucleotides formed from exogenous deoxycytidine are dUMP. The effect of tHU upon deoxycytidine incorporation was specific for herpes simplex virus-infected cells; THU did not increase deoxycytidine incorporation into DNA of uninfected cells. Therefore, one might expect THU to enhance the antiviral activity of 1-beta-D-arabinofuranasylcytosine since this analog is also readily deaminated. However, THU increased both the antiviral activity and the cell toxicity only slightly and to about the same extent. Therefore, the metabolism of 1-beta-D-arabnofuranosylcytosine is different from that of deoxycytidine in herpes simplex virus-infected cells.     

Inhibition of DNA polymerase from herpes simplex virus-infected wi-38 cells by phosphonoacetic Acid

Infection of Wi-38 cells with herpes simplex virus induced an elevated DNA polymerase activity which had many biochemical properties different from normal cell DNA polymerase. Phosphonoacetic acid specifically inhibited the virus-induced DNA polymerase as compared to the normal WI-38 cell DNA polymerase. The compound did not appear to inhibit enzyme activity by interacting with the DNA primer.     

Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine

We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 μM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.     

Efficacy of 4'-thio-5-ethyl-2'-deoxyuridine (TEDU) and its derivatives in experimental infection in mice caused by herpes simplex virus]

It was demonstrated that several 5'-phosphonates of 4'-thio-5-ethyl-2'-deoxyuridine possessed antiviral activity in vitro and in the murine model of herpes simplex virus type 1 infection. It was shown that the derivatives after intraabdominal administration penetrated effectively into the brain tissue. The agents provided statistically significant increase of the average life span, lower virus titre in the brain and lower lethality when compared to the control group of the animals. It is emphasized that the derivatives were less toxic than the original compound.     

Characterization of VP22 in herpes simplex virus-infected cells

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.     

New synthesis of 5-carboxy-2'-deoxyuridine and its incorporation into synthetic oligonucleotides

5-Carboxy-2'-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2'-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2'-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein

Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.     

Lifetimes of mRNA molecules directing the synthesis of viral proteins in herpes simplex virus-infected cells.

Possible variations in the functional lifetimes of herpes simplex virus type 1 mRNA molecules in infected HeLa cells were studied. As shown by the rate of decrease of radioactive amino acid incorporation into viral proteins after the addition of actinomycin, the average lifetime of early viral mRNA's are shorter than those for the late messenger species. In addition, when the viral proteins made after the addition of actinomycin were further analyzed by gel electrophoresis, it was found that messengers for individual viral proteins translated within the early or late time period also had some differences in their functional lifetimes. These results indicate that the synthesis of herpes simplex virus type 1 proteins during the replicative cycle is regulated in part by mechanisms controlling the functional lifetimes of viral mRNA's.     

Nuclear pore composition and gating in herpes simplex virus-infected cells

The mechanism by which herpes simplex virus (HSV) exits the nucleus remains a matter of controversy. The generally accepted route proposes that capsids exit via primary envelopment at the inner nuclear membrane and subsequent fusion of this primary particle with the outer nuclear membrane to gain capsid entry to the cytoplasm. However, recent observations indicate that HSV may induce gross morphological alterations of nuclear pores, resulting in the loss of normal pores and the appearance of dilated gaps in the nuclear membrane of up to several 100 nm. On this basis, it was proposed that a main route of capsid exit from the nucleus is directly through these altered pores. Here, we examine the biochemical composition of some of the major nuclear pore components in uninfected and HSV-infected cells. We show that total levels of major nucleoporins and their sedimentation patterns in density gradients remain largely unchanged up to 18 h after HSV infection. Some alteration in modification of one nucleoporin, Nup358/RanBP2, was observed during enrichment with anti-nucleoporin antibody and probing for O glycosylation. In addition, we examine functional gating within the nucleus in live cells, using microinjection of labeled dextran beads and a recombinant virus expressing GFP-VP16 to track the progress of infection. The nuclear permeability barrier for molecules bigger than 70 kDa remained intact throughout infection. Thus, in a functional assay in live cells, we find no evidence for gross perturbation to the gating of nuclear pores, although this might not exclude a small population of modified pores.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Contact-dependent interference with invariant NKT cell activation by herpes simplex virus-infected cells

Invariant CD1d-restricted NKT (iNKT) cells play important roles in generating protective immune responses against infections. In this study, we have investigated the role of human iNKT cells in HSV-1 infection and their interaction with epidermal keratinocytes. These cells express CD1d and are the primary target of the virus. Keratinocytes loaded with α-galactosyl ceramide (α-GalCer) could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer-dependent and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1-infected cells. Notably, CD1d downregulation was not observed on infected keratinocytes, which were also found to inhibit TCR-independent iNKT cell activation. Further examination of the cytokine profile of iNKT-keratinocyte cocultures showed inhibition of IFN-γ, IL-5, IL-10, IL-13, and IL-17 secretion but upregulation of IL-4 and TNF-α after the infection. Moreover, cell-to-cell contact between infected keratinocytes and iNKT cells was required for the inhibition of activation, as the cell-free supernatants containing virus did not affect activation. Productive infection of iNKT cells was however not required for the inhibitory effect. After coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer-loaded CD1d-expressing cells. We found that exposure to HSV-1-infected cells resulted in impaired TCR signaling downstream of ZAP70. Additionally, infected cells upregulated the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of iNKT cell responses was independent of galectin-9. Thus, interference with activation of human iNKT cells by HSV-1 may represent a novel immunoevasive strategy used by the virus to avoid immune clearance.     

Quantitation of the viral DNA present in cells transformed by UV-irradiated herpes simplex virus.

Two cell lines biochemically transformed by UV-irradiated herpes simplex virus (HSV) each contain virus DNA. A comparison of the kinetics of reassociation of 3H-labeled HSV DNA in the presence and absence of either clone 139 (HSV-1 transformed) or clone 207 (HSV-2 transformed) DNA showed that the presence of transformed cell DNA increased the rate of reassociation of approximately 10% of the viral genome while having no effect on the remaining 90%. The Cot1/2 of this reaction was approximately 1,000 in each cell type, as compared to approximately 3,000 for the cellular unique sequences. These results suggest the presence of four to six copies of a 10% fragment of the virus DNA per cell. The DNA from a hamster fibroblast cell line morphologically transformed by UV-irradiated HSV-2 (333-8-9) did not affect the rate of reassociation of HSV-2 DNA, indicating that these cells had less than 3% of a viral genome present.