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When restriction-active Escherichia coli cells (R+P1m+P1) are transformed with the pSF2124 plasmid, a common vector in experimental gene transfer, the efficiency of transformation (e.o.t.) is lowered by 2 orders of magnitude compared with restriction-negative (r-P1m-P1 or r-P1m+P1) recipient cells due to restriction of the pSF2124 DNA by endoR.EcoP1. Preinfection of r+P1m+P1 cells with UV-inactivated ocr+ phages (T3, T7, T3sam-) still able to express their early genes protects the plasmid DNA against restriction by endoR.EcoP1: The e.o.t. of r+P1m+P1 recipient cells with pSF2124 attains the same high value as that of r-m- cells. The specific role of the ocr+ gene function was demonstrated by the use of ocr- mutants (T3/R7, T7/D111): Preinfection with such phage mutants does not increase the e.o.t. of r+P1m+P1 cells. An unspecific e.o.t. alteration of restriction-negative (r-m-) recipient cells by ocr+ or ocr- phages was excluded. The ocr+ gene function can be exploited to protect pSF2124 against DNA restriction. The recipient cells survive the process of phage preinfection and transformation and stably replicate themselves as well as the plasmid DNA.

A number of novel 5-substituted 2'deoxypyrimidine nucleosides exhibited antiviral activity against herpes simplex virus type 1 strain V3 (HSV-1-V3) when assayed under one-step conditions in primary human lung fibroblast j(PHLF) cell cultures, and compared with the reference compounds cytosine arabinoside (ara-C), 5-iodo-2'-deoxyuridine (IUdR), and 5-iodo-5'amino-2',5'-dideoxyuridine (AIU). The most effective of these were (in order of decreasing activity): (E)-5-(2-bromovinyl)-UdR (BrVUdR) greater than ara-C greater than IUdR greater than 5-azidomethyl-UdR (AMeUdR) greater than 5-formyl-UdR (fUdR) greater than 5-hydroxymethyl-UdR (HMeUdR) greater than AIU greater than 5-mercaptomethyl-UdR (MMeUdR) = 5-hydroxymethyl-2'-deoxy-cytidine (HMeCdR) greater than 5-benzyloxymethyl-UdR (BOMeUdR). In a multistep virus replication experiment (plaque reduction assay on Vero cells) the order of decreasing activity was as follows: BrVUdR = ara-C greater than HMeUdR greater than fUdR IUdR greater than HMeCdR greater than BOMeUdR greater than AMeUdR greater than AIU greater than MMeUdR. BrVUdR effected a 50% reduction in plaque formation of different strains of HSV-1 at a concentration of 0.06-0.22 microM, of pseudorabies virus (PRV) at 0.02-0.23 microM, and of herpes simplex virus type 2 (HSV-2) at 8 microM, whereas the ID50 values for adenovirus type 2 and type 5 were 100 and 50-100 microM, respectively. The growth of synchronied baby hamster kidney cells in suspension cultures was inhibited by 50% at concentrations of 100, 70, 20, 4, 8, and 0.2 microM for BrVUdR, HMeCdR, IUdR, fUdR, BOMeUdR, and HMeUdR, respectively.

The temperature-sensitive DNA polymerase mutator mutants A58 and L98 are less inhibited by 3'-Fluorothymidine than the T4 wild type and the antimutator mutant CB121, in consequence of the assumed differences between the polymerase-associated exonuclease activities. This interpretation is confirmed by results with nalidixic acid and mitomycin C. The use of systems of different temperature-sensitive mutants of one or more genes is proposed for 1.) investigating the mode of action of drugs, 2.) studies on the mechanism of enzyme action and the functions affected in temperature-sensitive enzymes, and 3.) for enzyme-specific drug screening.

The intracellular growth of the phages T3 and T7 is restricted in the presence of the Escherichia coli prophage P1. Phage T3 has a higher ability to express its genome and to damage the host cell than T7. This partial protection of T3 against P1 restriction is due to the T3-coded SAMase, an enzyme which degrades S-adenosylmethionine, the cofactor of the P1 restriction endonuclease. Since we did not observe DNA cleavage in vivo, we conclude that the in vivo action of the P1 nuclease is limited to a SAM-dependent repressor-like binding to T3 and T7 DNA, while further reactions with the DNA (modification vs cleavage) are blocked.

The sugar-modified dTTP analogues 2',3'-didehydro-2',3'-dideoxy-thymidine 5'-triphosphate (ddeTTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), 3'-fluorothymidine 5'-triphosphate (FdTTP), and 3'-azidothymidine 5'-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID50 values of the compounds for the HIV-RT (ranging from 0.03 microM for ddeTTP to 0.1 microM for ddTTP) and the cellular DNA polymerase alpha (greater than 200 microM). DNA polymerase beta is partially affected by N3dTTP (ID50 = 31 microM) and by the other analogues (ID50 = 1-2.2 microM). FdTTP has proved as effective as N3dTTP (ID50 = 0.05 microM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 microM).

Nine ts mutants of pseudorabies virus (PRV) strain BUK were isolated following bromodeoxyuridine (BUdR) or NaNO2 mutagenesis by a selection technique based on a shiftdown from nonpermissive to permissive temperature. This technique is described and its advantages are discussed. The nine ts mutants have been assigned to four complementation groups.

Bovine leukaemia virus (BLV) and the human T-cell leukaemia/lymphoma viruses I and II represent a specific group of type-C RNA tumour viruses characterized by the presence between the err gene and the 3'LTR of an "x" region or LOR frame, which codes for a protein that trans-activates the transcription of the viral genome. As BLV can also infect sheep and induces pre B-cell specific tumours in these animals, we were interested in investigating whether suramin, a potent inhibitor of retrovirus-associated reverse transcriptase, may inhibit the in vivo multiplication of BLV in sheep. The sheep were infected with 4 X 10(7) leukocytes from a BLV-infected cow. The animals were maedi-visna virus-negative. Viral p24 antigen and reverse transcriptase appeared at 2 weeks and seroconversion occurred at 4 weeks after infection. Suramin was administered at 20 mg/kg/week from the 10th till the 16th week after infection. During the treatment period the expression of p24 antigen as well as the titre of anti-p24 and anti-gp51 antibodies were followed. Suramin treatment led to a significant, but transient, disappearance of p24 antigen and did not affect the titre of anti-p24 and anti-gp51 antibodies. The BLV-infected sheep may serve as a useful animal model for the investigation of retrovirus inhibitors and the evaluation of different therapeutic regimens.

In contrast to phage lambda the phages T3, T7 and T4 are not inhibited by as much as 150 microgram bleomycin/ml, while the chemically related antibiotic phleomycin increasingly inhibits the propagation of the phages in the order T4-T3-lambda. 20 microgram phleomycin/ml inhibit T3 by 95%. The resistance against bleomycin is surprising, because 10 microgram BM/ml block completely the colony-forming capacity of the host bacterium. The drug resistance of the phage growth correlates with the weak decrease of phage DNA synthesis, while the host cell DNA synthesis ceases rapidly. In accordance with these data is the in vivo inhibition of Escherichia coli cells and the in vitro degradation of their DNA. However, a contradiction exists between the in vivo resistance of T3 and T4 and the in vitro susceptibility of their DNA against nucleolytical fragmentation by bleomycin. The mechanism of the insensitivity of T3, T7 and T4 against bleomycin is unknown.

The permanently BLV-infected FLK cell line 44/2 and FLK sublines were tested for the stability of their BLV and antigen synthesis by three virus markers, p24, gp51, and activity of reverse transcriptase. The extent of BLV production in the FLK line correlated directly with the surface for cell growth in roller and stationary cultures. The synthesis and secretion of non-virus-associated gp51 is especially stimulated in the roller culture, and is largely independent of the quality of the culture medium. The roller culture allows considerable economy of the medium, at the same time retaining its full biological activity. As much as 2 mg of gp51 per litre of culture supernatant was obtained by this procedure. Appropriate markers for estimation of BLV production are p24 and gp51. For this purpose, the activity of reverse transcriptase on its own was not sufficient. The BLV yield differed by one order of magnitude among the 18 cloned FLK sublines. Three sublines have been identified, which showed a comparatively high virus production, under conditions of stationary cultures. The amount or activity of viral markers could not be correlated with the number of BLV proviruses.

We used purified bovine leukaemia virus (BLV) to test the inhibitory effects of suramin, HPA-23, and 3'-azidothymidine triphosphate (N3dTTP) on the reverse transcriptase activity. The 50% inhibitory concentrations (ID50) of the compounds were determined to be 2.8 mumol/l (suramin), 8.0 mumol/l (HPA-23), and 0.17 mumol/l (N3dTTP). Kinetic analyses of suramin and HPA-23 inhibition are discussed. The observed inhibitory effects emphasize the suitability of BLV as a model virus for investigations of retrovirus chemotherapy.

Certain differences between phage T3 on the one hand and T3sam- and T7 on the other hand indicate that the T3-coded SAMase function is responsible (i) for the development of the pseudolysogenic state by preventing T3 DNA methylation, and (ii) for the partial protection of the phage DNA against restriction by the P system.

Calves were immunised by means of a native Al(OH)3-absorbed gp51-preparation which had been obtained from a foetal lamb kidney cell line, following bovine leucosis virus (BLV) infection. 9 animals were immunised 3 times, using 300 micrograms gp51. 7 animals underwent test infection, using 2.5 x 10(3) or 2.5 x 10(4) BLV-infected lymphocytes. Serological and virological reactions of all animals, including 3 calves which had received only test infections, were followed up through 40 weeks by means of immunodiffusion test, enzyme-immuno-assay, gp51 antibody radio-immuno-assay, reverse transcriptase test, syncytial test, competitive p24 radio-immuno-assay, and by transmission of whole blood in animal experiments. The results obtained from virological testing showed that 1 animal had been protected by preceding immunisation. 4 in 7 immunised and test-infected animals exhibited transient BLV values, between the 7th and 16th weeks from infection. Typical leucosis infection had been induced to 2 animals. The above findings are discussed and are compared to similar results recorded by other working groups.

The ability of the bacteriophage T3 to adsorb on the host cells of Escherichia coli W1655 changes depending on the host strain in which the phage was propagated before. This phenomenon is termed "non-classical" host-controlled modification in contrast to "classical" DNA modification. We demonstrate here that T3 phages with various non-classical modifications as well as the host range mutant T3hW differ from each other in the antigenic determinants of the phage adsorption protein.

The bacteriophages T3 and T7 are not modified and restricted by E. coli strains with different host specificity (E. coli B, K, O) in vivo. The phages code for a gene product with the ability to overcome classical restriction (ocr): ocr- mutants are subject to modification and restriction via DNA methylation vs cleavage. The T3 genome possesses recognition sites for the restriction endonuclease R.EcoB which, unless the DNA is B-specifically modified, trigger 5-7 DNA cleavages. The ocr gene function of T3 and T7 is located within the gene 0.3 region of these phages and is not identical with the sam (SAMase) function of T3. The mechanism of ocr protection remains unclear, while it is certain that this protection by the gene 0.3 protein is exerted in the infected cell and not through "over-all" modification in the preceding growth cycle of the phage.

By means of SDS PAGE we isolated from virus-infected foetal lamb kidney (FLK) cells a relatively homogenous envelope transmembrane protein gp30 of bovine leukaemia virus (BLV). As shown by a partial sequence analysis of the N-terminus of this protein, our gp30 preparation contained only traces (less than 5%) of p24 gag protein: Rabbit anti-gp30 serum did not cross react with the BLV proteins gp51, p12, p15(1), p15(2), and p10 but reacted weakly with the p24 polypeptide. 125I-labelled gp30 (chloramine-T) was precipitated with the serum of BLV-infected cattle. Nonlabelled preparation of gp30 competitively inhibited the reaction of 125I-labelled gp30 with the natural antibodies. We investigated 193 cattle sera by liquid phase radioimmunoassays (RIA) using 125I-gp30, gp51 and p24 antigens. Sixteen noninfected cattle sera were negative in all tests. The 177 serum samples of BLV-infected animals were examined to the diagnostic value of the three tests. Of these, 175 were positive in gp51 RIA, 172 in p24 RIA and 164 in gp30 RIA. In all three tests, 159 sera were positive while 18 sera, mostly coming from animals with normal leukocyte counts, were positive only either with gp51 or p24, or were double positive with either gp51/p24 or gp51/gp30. We conclude that the gp51 RIA is superior to both the gp30 and the p24 RIA and that the gp30 RIA will be useful for investigating the role of gp30 in virus pathogenicity.

(E)-5-(2-Bromovinyl-2'-deoxyuridine (BrVUdR) showed strong antiviral activity against different laboratory strains and clinical isolates of herpes simplex virus type 1 (HSV-1) on primary rabbit testes (PRT) cells with a 50% inhibition of plaque formation (ID50) at 0.01-0.02 microM. One laboratory strain (HSV-1-S), however, was completely refractory even at concentrations as high as 100 microM. In contrast, the ID50S for all herpes simplex virus type 2 (HSV-2) strains were about 10(2) - 10(3) times higher (8-25 microM) than for the HSV-1 strains. No toxicity in mice treated with 140 mg BrVUdR/kg/day for 14 days was observed, and successful treatments of herpes encephalitis in mice induced experimentally by intracerebral infection with one laboratory strain (HSV-1-Kupka) and one clinical isolate (HSV-1-64) were achieved. Treatment of encephalitis in mice induced by the strain HSV-1-S insensitive to BrVUdR in cell culture failed to be effective. Similar antibody titers against HSV-1 were found in surviving mice of the control and of the BrVUdR-treated groups.

In an enzyme-specific drug screening system nalidixic acid and 3'-FTdR, inhibitors of DNA synthesis, both reduce the growth of wild type and temperature-sensitive point mutants of phage T3 with different efficiencies. The wild type shows the strongest sensitivity against the drugs, while an exonuclease mutant is the most insensitive variant. The DNA polymerase mutants exhibit an intermediate degree of inhibition. The anthracycline antibiotics violamycin BI and adriblastin which preferentially inhibit RNA synthesis show the same degree of inhibition for all mutants. This is true also for the RNA synthesis inhibitor lambdamycin, which is identical with chartreusin. The protein synthesis inhibitors chloramphenicol and o-phenanthroline, a chelating agent, impair all mutants to the same extent. Our data confirm the hypothesis that structural variants of essential viral enzymes, when compared with the wild type should reveal different sensitivities against specific inhibitors and show that this T3 system could be used for the indication of specific inhibitors of DNA synthesis.

The closely related phages T3 and T7 exhibit different growth patterns on Escherichia coli W hosts cells (E. coli K12 derivatives). T7 grows normally while T3 does not adsorb. T3hw mutants displaying a T7-like host range were isolated and described.

The growth properties of pseudorabies virus (PRV) strain V BUK, wild type and its four ts mutants, were examined using highly accurate temperature measurements in the incubator and on the cell sheet. The transition from permissive to nonpermissive temperature occurred within an interval of 1 degree C. The midpoint of this interval was termed the semipermissive temperature since at this temperature the reproduction and consequently the plaquing of mutant viruses was not completely restricted. The "leakiness" of ts mutants might be due to their incubation at semipermissive temperature. Under semipermissive conditions and at high virus inputs, three mutants exhibited nearly complete inhibition of plaquing. In an experimental mixture of to mutants and PRV-BUK wild type, the latter was not affected by the inhibition. This might be due to an enhanced sensitivity of the mutants, but not of the wild type, to interferon under these conditions.

Over 90% of the total viral information together with the adjacent 5' cellular sequences were cloned in the lambdoid spi selection vector lambda-2558 by taking advantage of the unique EcoRI restriction site very close to the 3' long terminal repeat. Of the fifteen isolated recombinant phages with viral inserts, one has been propagated and its DNA isolated and subjected to preliminary restriction endonuclease analysis. The proviral insert has been found to be almost identical to the unintegrated proviral DNA reported by Kashmiri et al. (1984).

In view of the coincidence of antiviral and antiparkinsonism activities of amantadine four antiparkinsonism drugs, NorakinR (triperiden), ParkopanR (trihexyphenidyl), AntiparkinR (diethylbenzhydramine) and AkinetonR (biperiden) were tested for antiviral activity in various virus-cell systems. Norakin inhibited the replication of influenza A viruses in chick embryo fibroblast, MDCK and Ehrlich ascites tumour cells. It also inhibited the replication of measles virus in Vero cells, 50% inhibitory concentrations being 2-6 micrograms/ml. The drugs were also active against influenza B virus. Several representatives of other virus families, e.g. vaccinia, vesicular stomatitis, polio type 1 and herpes simplex type 1 viruses were insensitive to the compounds.

3'-fluorothymidine (FddThd) is well phosphorylated to the 5'-triphosphate in various relevant cell-lines. This results in fairly stable levels of this compound without accumulation of the 5'-monophosphate to the extent described for 3'-azidothymidine (AzT). The di- and triphosphate of FddThd seems unable to influence the ribonucleotide reductase in permeable cells. FddThd protects 50% of the MT-4 cells against the cytopathic effect of HIV at 3 nM, but reduces the number of uninfected viable cells to 50% at 1.1 microM. All other tested human cell-lines displayed a far less antiproliferative sensitivity to FddThd, comparable with that of AzT.

Foreign F'lac plasmid DNA which is introduced into potentially restricting E. coli recipient cells can be protected from restriction by preinfecting the recipient cells with UV-inactivated T3 or T7 bacteriophages which express the ocr gene function. The recipient cells survive and are able to replicate themselves as well as the newly acquired plasmid.