2-Amino-6-arylsulfonylbenzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1.

A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.

Deletions in the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase are observed in HIV-1 isolated from subjects during long-term antiretroviral therapy.

OBJECTIVES: To examine the effect of in-frame deletions in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on plasma viremia and phenotypic resistance to antiretroviral drugs. STUDY DESIGN/METHODS: Plasma HIV-1 RNA was isolated from 168 antiretroviral therapy-experienced subjects for quantification of plasma viremia, RT sequence analysis, and phenotypic resistance assays. RESULTS: Four patients were found to harbor HIV-1 strains possessing in-frame, 3-nucleotide deletions at RT codons 67, 69, and 70. In these subjects, phenotypic resistance and high plasma viremia were observed only in a background of multiple resistance mutations. A recombinant virus engineered with an in-frame deletion of RT codon 67 did not have increased resistance to nucleoside reverse transcriptase inhibitors (NRTIs). CONCLUSIONS: Selection for deletions within the beta3-beta4 hairpin loop of the HIV-1 RT is an uncommon event most likely to occur in subjects with long-term antiretroviral experience. The codon 67 deletion does not appear to cause increased phenotypic resistance or increased viremia in the absence of concomitant RT mutations.

Characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.

Permeabilized preparations of equine infectious anemia virus (EIAV) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral DNA. When (-) and (+) strand products were analyzed by Southern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site for initiation of DNA synthesis and a specific mechanism of (+) strand termination. Precise localization of this (+) strand origin within the integrase (IN) coding region was achieved through its in vitro selection and extension into, and excision from, nascent DNA by purified recombinant p66/p51 EIAV reverse transcriptase (RT), suggesting that the EIAV genome harbors a central polypurine tract (cPPT). In addition, a model system was developed for evaluating whether sequences immediately downstream of the cPPT would terminate (+) strand synthesis in the context of strand displacement. Such a sequence was indeed discovered which functions in a manner analogous to that of the central termination sequence (CTS) of HIV, where A-tract-induced minor groove compression has been suggested to induce localized distortion of the nucleic acid duplex and termination of (+) strand synthesis. This interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine for adenine, thereby relieving minor groove compression. The nucleotide substitution can also shift the site of termination in strand displacement (+) strand synthesis. Collectively, our data support proposals that lentiviruses may have evolved specialized mechanisms for initiating and terminating (+) strand DNA synthesis at the center of their genomes.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol.

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a human immunodeficiency virus-1 in vitro DNA synthesis system to study reverse transcriptase inhibitors.

A Human immunodeficiency virus type-1 endogenous reverse transcriptase reaction was developed as an in vitro assay to study the inhibition of reverse transcription by antiviral compounds. Conditions were established for producing genomic length (-) strand DNA in high yields and measuring the inhibition of this transcript as the assay endpoint. In addition to genomic length (-) strand DNA, a novel segmented (-) strand product composed of a 6.0 kb reverse transcript of the 5' 2/3 of the viral RNA genome and a 3.5 kb reverse transcript of the 3' 1/3 was observed. The most prominent (+) strand product was the size expected for plus-strong stop DNA. Additional minor (+) strand species were also observed. The triphosphate form of the nucleoside analog inhibitor 3'-azido-3'-deoxythymidine (RETROVIR, Zidovudine, AZT) and BI-RG-587 (nevirapine), a non nucleoside inhibitor, were used to demonstrate the utility of the endogenous system for the analysis of reverse transcriptase inhibitors. In a standard reaction, synthesis of genomic length DNA was 50% inhibited by 0.1 microM AZTTP and 0.1 microM nevirapine.

Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction.

The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecular-weight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.

Analysis of Fv-1 restriction in two murine embryonal carcinoma cell lines and a series of differentiated derivatives.

We have used antibiotic-resistant retrovirus vectors rescued by Fv-1-sensitive murine leukaemia viruses (MuLV) to examine the Fv-1 phenotype of two undifferentiated embryonal carcinoma (EC) cell lines derived from teratocarcinomas of mouse strain 129. In addition, a set of EC cell-derived differentiated cell lines was analysed. Restriction of both B-tropic and endogenous N-tropic virus is characteristic of the Nr-type restriction reported in mouse strain 129. However, results indicate that Fv-1 restriction is not expressed in the PCC4.aza1R EC cell line. In contrast, the F9 EC cell line showed a strong restriction of the B-tropic pseudotyped vector but failed to restrict endogenous N-tropic pseudotypes. The Fv-1 gene thus seems to be differentially expressed in two EC cell lines derived from the same mouse strain. Furthermore, the selective restriction of B-tropic but not endogenous N-tropic MuLV in F9 cells suggests that these activities function independently of each other. Analysis of PCC4.aza1R-derived differentiated cell lines revealed that three fibroblast cell lines derived by retinoic acid-induced differentiation were also phenotypically silent for Fv-1. However, a pre-adipocyte line established following simultaneous exposure to retinoic acid and 5-azacytidine showed strong restriction of both B-tropic and endogenous N-tropic MuLV. Although additional data suggest that there is no correlation between the differentiated pre-adipocyte phenotype and Fv-1 expression, our results nonetheless show that Nr restriction can be observed in some derivatives of PCC4.aza1R cells, presumably by activating expression of the Fv-1 gene.

Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line.

The Fv-1b-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the Fv-1b restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genome-directed processes.

Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors.

We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.

Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells.

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.

Increased cholesterol sulfate and cholesterol sulfotransferase activity in relation to the multi-step process of differentiation in human epidermal keratinocytes.

In this study the synthesis of cholesterol sulfate is examined in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK) in culture. During the exponential growth phase, NHEK cells exhibit a relatively high colony-forming efficiency and appear undifferentiated on the basis of their morphology and expression of biochemical characteristics. At confluence, the cells undergo terminal differentiation that is characterized by the commitment to terminal cell division (reduction in colony-forming ability) and expression of the differentiated phenotype. An accumulation of cholesterol sulfate accompanies this program of differentiation. This accumulation of cholesterol sulfate parallels the increase in transglutaminase type I activity and the competence to form cross-linked envelopes, whereas it precedes the "spontaneous" formation of cross-linked envelopes. Increased cholesterol sulfotransferase activity appears to account for the increase in cholesterol sulfate. The cholesterol sulfate accumulation, as well as the increase in cholesterol sulfotransferase and transglutaminase activity, are inhibited by retinoids. However, the presence of retinoids does not prevent NHEK cells from undergoing terminal cell division at confluence. Two NHEK cell lines expressing SV40-large T antigen also undergo terminal differentiation at confluence and start to accumulate cholesterol sulfate. Two other, differentiation-defective cell lines do not exhibit an increase in cholesterol sulfate at confluence. These results show that epidermal keratinocytes in culture, like cells in the epidermis, accumulate cholesterol sulfate when undergoing squamous differentiation. This program appears to consist of a retinoid-insensitive step (commitment to terminal cell division) and a retinoid-sensitive step (expression of the squamous differentiated phenotype).

Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.

Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp gag-pol sequence with ACTGCCC repeat, a 113-bp mid-pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse species showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus, all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes.

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.

Characterization of two solitary long terminal repeats of murine leukemia virus type that are conserved in the chromosome of laboratory inbred mouse strains.

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage lambda. Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites. Two of the three subclones were found to be identical except for their orientations in the vector pBR322. Unique-sequence regions on either side of the two nonidentical elements were used to characterize their integration sites in genomic DNA. The solitary LTRs and their flanking regions were found to be conserved in a number of inbred mouse strains, including three strains known not to harbor endogenous ecotropic MuLV-type proviruses. Comparison of cleavage by the methylation-sensitive restriction enzyme SmaI and methylation-insensitive KpnI at the characteristic LTR SmaI/KpnI site suggested that at least one of these solitary LTRs is methylated to a lesser extent than are most endogenous proviral LTRs. These particular solitary LTRs, like endogenous proviral sequences, appear to be stably transmitted genetic elements.

Activation of the K-ras protooncogene in lung tumors from rats and mice chronically exposed to tetranitromethane.

Dominant transforming genes were detected in lung tumors from Fischer 344 rats and C57BL/6 X C3H F1 mice chronically exposed by inhalation to tetranitromethane, a highly volatile compound used in several industrial processes. The rat lung neoplasms were classified as adenocarcinomas, squamous cell carcinomas (epidermoid carcinomas), or adenosquamous carcinomas. The mouse lung tumors were classified as papillary adenocarcinomas or adenomas. In both species, the tumors were morphologically similar to lung tumors in humans. The transfection assay using NIH/3T3 mouse fibroblasts detected transforming genes in 74% (14 of 19) of the rat lung tumors and in 100% (4 of 4) of the mouse lung tumors. Southern blot analysis indicated that transforming gene was an activated K-ras protooncogene in both species. The first exon of the K-ras gene in normal DNA and in DNA from two cell lines transformed by tumor DNA was compared by cloning and sequencing the gene. Experiments showed that there was a GC----AT transition in the second base of the 12th codon of the K-ras oncogene in the two transfectant DNAs. Oligonucleotide hybridization indicated that all of the rat and mouse transfectants had this activating lesion. Additional tumor DNA was then tested for the presence of a mutated allele with the GC----AT transition. All of the rat tumors tested and all of the mouse tumors tested had this mutation present. Hybridization using the normal oligonucleotide sequence around the 12th codon indicated that the normal allele was also present in the majority of the tumors, suggesting that the loss of normal allele is not necessary for the development of neoplasia. One rat lung tumor had no normal allele present, possibly suggesting that this tumor could have been in a more advanced stage than the other tumors. This is the first study to detect activated protooncogenes in rodent tumors induced under conditions which mimic human exposure to a chemical in the workplace. Tetranitromethane may exert its carcinogenic action by both activation of the K-ras oncogene and stimulation of cell proliferation by its irritant properties.

Characterization of a molecular clone of RFM/Un mouse chromosomal DNA that contains a full-length endogenous murine leukaemia virus-related proviral genome.

A 12.4 kbp HindIII chromosomal DNA fragment harbouring an apparently intact 9.2 kbp endogenous murine leukaemia virus (MuLV)-related proviral genome was isolated from an RFM/Un strain mouse by molecular cloning and designated pRFM #6. Nucleotide sequence analysis revealed the following characteristic features in the pRFM #6 provirus: a distinct 200 bp sequence in the long terminal repeat (LTR) mid-U3 region, a primer binding site for glutamine tRNA, a 3' pol region encoding an 'endonuclease' protein of 390 amino acids, and the mink cell focus-forming virus type-specific sequence at the 5' portion of the env gene. The 699 bp 5' LTR and 700 bp 3' LTR of pRFM #6 provirus were identical except for three base changes in the U3 'enhancer' region. At the cell-provirus DNA junction, 4 bp direct repeats were present. The proviral genome was found at the same chromosomal DNA site in BALB/c, AKR, C3H, CBA and RFM strain mice, but not in NFS/N or C57BL/6 strain mice.

Hematopoietic neoplasias of the RFM/Un mouse contain somatic re-integration of the restriction endogenous ecotropic provirus.

We have examined the spleen DNA of individual mice of the RFM/Un strain for evidence of re-integration of the endogenous ecotropic provirus in radiation-induced and spontaneous neoplasms. The ecotropic env specific probe detects only a single 19 kb EcoRI or a single 7.0 kb HindIII fragment in all DNA preparations from normal tissues of RFM mice, corresponding to the endogenous provirus. Additional DNA restriction fragments containing the ecotropic virus (eco) specific sequence, corresponding to somatically acquired provirus, are detected in two out of five spleen DNA samples from animals with myeloid leukemia and one of three with thymic lymphoma. In addition somatically acquired eco-specific fragments are also detected in greater than 85% of DNA samples from reticulum cell sarcomas, a late occurring spontaneous hematopoietic neoplasm in this mouse strain. These results are consistent with a 'promoter/enhancer insertion' model of leukemogenesis involving the endogenous ecotropic provirus and are of particular interest since the RFM/Un mouse possesses a locus that restricts exogenous infection of cells by the endogenous virus.

Nucleotide sequence analysis of endogenous murine leukemia virus-related proviral clones reveals primer-binding sites for glutamine tRNA.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.