The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50µM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 µM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

4-Thiouridine-Enhanced Peroxidase-Generated Biotinylation of RNA.

Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-thiouridine (s4 U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4 U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4 U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.

Local administration of 4-Thiouridine, a novel molecule with potent anti-inflammatory properties, protects against experimental colitis and arthritis.

Previous studies in a rat model of Sephadex induced lung inflammation showed that 4-Thiouridine (4SU), a thiol substituted nucleoside, was very effective in reducing edema, leukocyte influx and TNF levels in bronchoalvelolar lavage fluid. However, little is known about the factors and mechanisms underlying these effects. In the present study, we have used two separate mouse models of chronic inflammation, a model of dextran sulphate sodium (DSS) induced colitis and a model of antigen induced arthritis, to evaluate the anti-inflammatory effect of 4-thiouridine. We have analyzed a broad spectrum of inflammatory mediators in order to delineate the mechanisms behind a potential anti-inflammatory effect of 4SU. Colitis was induced in C57BL/6 mice by administration of 3.5% DSS in drinking water for 5 days and the potential anti-colitic effect of 4SU was assessed by monitoring the disease activity index (DAI), measurement of colon length and histopathological analysis of colon tissue. We analyzed tissue myeloperoxidase (MPO) activity, serum pro-inflammatory cytokines (IL-1ß, IL-6 and TNF), mRNA and protein expression of pro-inflammatory cytokines, COX-2, and NF-κB activity in colitis tissue. Intracolonic administration of 4SU (5 mg/kg & 10 mg/kg.) significantly inhibited MPO activity and reduced the levels of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF) as well as COX-2. Further, NF-κB activation was also blocked by attenuating the phosphorylation of IkB kinase (IKK α/ß) in DSS-induced colitis tissues. Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA and 4SU was administered locally by direct injection into the knee joint. The antiarthritic potential of 4SU was calculated by histopathological scores and histochemical analysis of joint tissue. Further, immunohistochemistry was used to study inflammatory cell infiltration and expression of cytokines and adhesion molecules in the synovium. Local administration of 50-100 mg/kg 4SU at the time of arthritis onset clearly prevented development of joint inflammation and efficiently inhibited synovial expression of CD18, local cytokine production and recruitment of leukocytes to the synovium. Taken together, our data clearly demonstrates a potent anti-inflammatory effect of 4SU in two experimental models. In conclusion 4SU could be a new promising candidate for therapeutic modulation of chronic inflammatory diseases like ulcerative colitis and arthritis.

Why Does the Type of Halogen Atom Matter for the Radiosensitizing Properties of 5-Halogen Substituted 4-Thio-2'-Deoxyuridines?

Radiosensitizing properties of substituted uridines are of great importance for radiotherapy. Very recently, we confirmed 5-iodo-4-thio-2'-deoxyuridine (ISdU) as an efficient agent, increasing the extent of tumor cell killing with ionizing radiation. To our surprise, a similar derivative of 4-thio-2'-deoxyuridine, 5-bromo-4-thio-2'-deoxyuridine (BrSdU), does not show radiosensitizing properties at all. In order to explain this remarkable difference, we carried out a radiolytic (stationary and pulse) and quantum chemical studies, which allowed the pathways to all radioproducts to be rationalized. In contrast to ISdU solutions, where radiolysis leads to 4-thio-2'-deoxyuridine and its dimer, no dissociative electron attachment (DEA) products were observed for BrSdU. This observation seems to explain the lack of radiosensitizing properties of BrSdU since the efficient formation of the uridine-5-yl radical, induced by electron attachment to the modified nucleoside, is suggested to be an indispensable attribute of radiosensitizing uridines. A larger activation barrier for DEA in BrSdU, as compared to ISdU, is probably responsible for the closure of DEA channel in the former system. Indeed, besides DEA, the XSdU anions may undergo competitive protonation, which makes the release of X- kinetically forbidden.

5-Iodo-4-thio-2'-Deoxyuridine as a Sensitizer of X-ray Induced Cancer Cell Killing.

Nucleosides, especially pyrimidines modified in the C5-position, can act as radiosensitizers via a mechanism that involves their enzymatic triphosphorylation, incorporation into DNA, and a subsequent dissociative electron attachment (DEA) process. In this paper, we report 5-iodo-4-thio-2'-deoxyuridine (ISdU) as a compound that can effectively lead to ionizing radiation (IR)-induced cellular death, which is proven by a clonogenic assay. The test revealed that the survival of cells, pre-treated with 10 or 100 µM solution of ISdU and exposed to 0.5 Gy of IR, was reduced from 78.4% (for non-treated culture) to 67.7% and to 59.8%, respectively. For a somewhat higher dose of 1 Gy, the surviving fraction was reduced from 68.2% to 54.9% and to 40.8% for incubation with 10 or 100 µM ISdU, respectively. The cytometric analysis of histone H2A.X phosphorylation showed that the radiosensitizing effect of ISdU was associated, at least in part, with the formation of double-strand breaks. Moreover, the cytotoxic test against the MCF-7 breast cancer cell line and human dermal fibroblasts (HDFa line) confirmed low cytotoxic activity of ISdU. Based on the results of steady state radiolysis of ISdU with a dose of 140 Gy and quantum chemical calculations explaining the origin of the MS detected radioproducts, the molecular mechanism of sensitization by ISdU was proposed. In conclusion, we found ISdU to be a potential radiosensitizer that could improve anticancer radiotherapy.

2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation.

BACKGROUD: BST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface. RESULTS: Using a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and ß-TrCP2, but decreases BST-2 ubiquitination. CONCLUSION: Our study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.

4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response.

High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments.

A forward-genetic screen and dynamic analysis of lambda phage host-dependencies reveals an extensive interaction network and a new anti-viral strategy.

Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli) genes--over half of which have not been previously associated with infection--that when knocked out inhibited lambda phage's ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation-one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNAGlu, tRNAGln, and tRNALys is particularly important for the efficient production of infectious lambda phage particles.

5-Nitro-2-pyridyl-1-thioglucosides: application in synthesis of analogues of glycosyltransferases natural substrates.

5-Nitro-2-pyridyl-1-thioglucosides were used in synthesis of complex uridine derivatives (13-16) in two different sequences of reactions. In one route, the first step was glycosylation of selectively protected 5-nitro-2-pyridyl-1-thioglucoside 1 with two different glycosyl donors (5 or 6), next, the nitro group in aglycone of obtained disaccharides 7 or 8 was reduced and then obtained products 9 or 10 were condensed with uridine derivatives 3 or 4 using DMT-MM as condensing agent under microwave irradiation. In the second route, condensation and glycosylation reactions were applied in reverse order. As it turned up, a sequence of reactions affected the yield of final glycoconjugates 13-16 and depended on the type of uridine derivatives used.

Antiproliferative effect of 4-thiouridylate on OCM-1 uveal melanoma cells.

PURPOSE: Brachytherapy is a well-established, effective treatment for uveal melanoma with a failure rate of 15%. The fatal consequence of unsuccessful treatments offers reason for improvement of the method. The authors propose using an apoptosis inducing agent locally, concomitantly with the well-established therapy, to sensitize the tumor cells. The authors propose a new nontoxic moderately active apoptosis inducing agent, 4-thio-uridylate (s4UMP), for this purpose. METHODS: OCM-1 uveal melanoma cells were treated with various concentrations of s4UMP and its effect was monitored by measuring the cell viability (MTT assay). The following apoptosis detecting methods were performed to reveal the mechanism of decreased cell viability: light microscopy, DNA fragmentation assay, determination of caspase 9 activity, and FACS analysis. RESULTS: The viability of uveal melanoma cells was decreased by 32%, 40%, and 9% after 24, 48, and 72 hours of treatment with 10 microg/mL (30 microM) s4UMP. The effect was not dose dependent; it rather followed a saturation-type inhibition and the cells at lower drug concentration recovered after 72 hours. Characteristic apoptotic cell morphology and DNA fragmentation was detected in treated cells. The caspase-9 was activated upon treatment showing maximal activity at 48 hours suggesting the induction of apoptosis. The annexin binding activity further verified the apoptogenic activity of s4UMP. CONCLUSIONS: Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

Effects of uridine, isomatitol and 4-thiouridine on in vitro cell adhesion and in vivo effects of 4-thiouridine in a lung inflammation model.

Since leukocyte adhesion to endothelial cells is crucial for extravasation of leukocytes to sites of inflammation, inhibition of cell-cell adhesion has been suggested as a means to achieve selective modulation of the immune system. We have, using a static in vitro adhesion assay involving adhesion of granulocytes to tumor necrosis factor alpha (TNFalpha)-stimulated human umbilical vein endothelial cells (HUVEC), found three substances--uridine, isomaltitol and 4-thiouridine-that, independently and significantly, reduced leukocyte adhesion by approximately 30-65%. 4-Thiouridine was also tested in an in vivo model of Sephadex (SDX)-induced lung inflammation with Sprague-Dawley rats. Intratracheal instillation of Sephadex (5 mg/kg) alone resulted in a dramatic increase in lung edema and total leukocyte count after 24 h. A differential count of bronchoalveolar lavage (BAL) cells indicated an increased influx of macrophages, eosinophils and neutrophils. Co-administration of 4-thiouridine significantly reduced lung edema by 38%. There was also a significant reduction of the total leukocyte count by 58%. The differential leukocyte count indicated that eosinophil influx alone was reduced by 70%. After Sephadex challenge, we found elevated levels of TNFalpha--an important inflammatory mediator--in the bronchoalveolar lavage fluid (BALF). TNFalpha levels were significantly reduced by more than 80% by co-administration of 4-thoiuridine. These results suggest that uridine, isomaltitol and, especially, 4-thiouridine affect adhesion between leukocytes and activated endothelium, and warrant further in vitro and in vivo studies.

4-Thio-5-bromo-2'-deoxyuridine: chemical synthesis and therapeutic potential of UVA-induced DNA damage.

4-Thio-5-bromo-2'-deoxyuridine (3a) is prepared from 5-bromo-2'-deoxyuridine (BrdU) and its key properties are explored. The thionucleoside (3a) can react readily with monobromobimane and produces high fluorescence. 3a has UV maximum absorption at 340 nm and can be incorporated into cellular DNA. The cells containing 3a become sensitive to UVA light, offering therapeutic potential for UVA-induced cell killing.

Pyrimidinethione nucleosides and their deaza analogues.

The methods of preparation, structure, chemical properties and synthetic potentiality of pyrimidinethione nucleosides and their deaza analogues are reported.

4-thio-U cross-linking identifies the active site of the VS ribozyme.

To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4-thio- uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross-linked RNAs, in which the 4-thio-uridine was cross-linked to A756 in the 730 loop of helix VI. The cross-links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross-linked RNAs contains a lariat, indicative of intramolecular cross-linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme-substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix I interacts with the 730 loop in helix VI.

Identification of active antiviral compounds against a New York isolate of West Nile virus.

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies for this disease. A chemotherapeutic approach may be a reasonable strategy because the virus infection is typically not chronic and antiviral drugs have been identified to be effective in vitro against other flaviviruses. A panel of 34 substances was tested against infection of a recent New York isolate of WNV in Vero cells and active compounds were also evaluated in MA-104 cells. Some of these compounds were also evaluated in Vero cells against the 1937 Uganda isolate of the WNV. Six compounds were identified to be effective against virus-induced CPE with 50% effective concentrations (EC50) less than 10 microg/ml and with a selectivity index (SI) of greater than 10. Known inhibitors of orotidine monophosphate decarboxylase and inosine monophosphate dehydrogenase involved in the synthesis of GTP, UTP, and TTP were most effective. The compounds 6-azauridine, 6-azauridine triacetate, cyclopententylcytosine (CPE-C), mycophenolic acid and pyrazofurin appeared to have the greatest activities against the New York isolate, followed by 2-thio-6-azauridine. Anti-WNV activity of 6-azauridine was confirmed by virus yield reduction assay when the assay was performed 2 days after initial infection in Vero cells. The neutral red assay mean EC50 of ribavirin was only 106 microg/ml with a mean SI of 9.4 against the New York isolate and only slightly more effective against the Uganda isolate. There were some differences in the drug sensitivities of the New York and Uganda isolates, but when comparisons were made by categorizing drugs according to their modes of action, similarities of activities between the two isolates were identified.

Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift.

Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells. In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins. This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site. Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift. The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function.

Synthesis and hypnotic activities of 4-thio analogues of N3-substituted uridines.

Reaction of tri-O-acetyluridine (1) with benzyl bromide or 2-chloroacetophenone in the presence of K2CO3 gave the N3-substituted analogues 2a,c. Condensation of 1 with (+/-)-1-phenylethanol or 3,5-dimethylbenzyl alcohol using the Mitsunobu reaction also gave 2b,d in good yields. These compounds were allowed to react with Lawesson's reagent and were subsequently treated with ammonia to afford the 4-thiouracil derivatives 5a-d. Compounds 5a-c showed moderate hypnotic activity in mice. However, N3-(3,5-dimethyl)benzyl derivatives 3d, 5d were found to be almost inactive in this assay.

Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase.

The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-beta-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil].

Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking.

The 16S rRNA central pseudoknot region in the 30S ribosomal subunit has been investigated by photocrosslinking from 4-thiouridine (s4U) located in the first 20 nt of the 16S rRNA. RNA fragments (nt 1-20) were made by in vitro transcription to incorporate s4U at every uridine position or were made by chemical synthesis to incorporate s4U into one of the uridine positions at +5, +14, +17, or +20. These were ligated to RNA containing nt 21-1542 of the 16S rRNA sequence and, after gel purification, the ligated RNA was reconstituted into 30S subunits. Long-range intramolecular crosslinks were produced by near-UV irradiation; these were separated by gel electrophoresis and analyzed by reverse transcription reactions. A number of crosslinks are made in each of the constructs, which must reflect the structural flexibility or conformational heterogeneity in this part of the 30S subunit. All of the constructs show crosslinking to the 559-562, 570-571, and 1080-1082 regions; however, other sites are crosslinked specifically from each s4U position. The most distinctive crosslinking sites are: 341-343 and 911-917 for s4U(+5); 903-904 (very strong), 1390-1397, and 1492 for s4U(+14); and 903-904 (moderate) for s4U(+17); in the 1070-1170 region in which there are different patterns for each s4U position. These results indicate that part of the central pseudoknot is in close contact with the decoding region, with helix 27 in the 885-912 interval and with part of domain III RNA. Crosslinking between s4U(+14) and 1395-1397 is consistent with base pairing at U14-A1398.

Anti-(herpes simplex virus) activity of 4'-thio-2'-deoxyuridines: a biochemical investigation for viral and cellular target enzymes.

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.