Molecular cloning of AKR xenotropic murine leukemia virus unintegrated proviral DNA

Suprahelical proviral DNA of AKR xenotropic murine leukemia virus was purified from agarose gels and cloned in lambda Charon 28 DNA (BamHI sites). Nine viral DNA recombinants were identified and mapped with 12 restriction endonucleases. Three calsses of cloned viral DNA inserts were found: (1) Six inserts were apparently full-length 9.0-kb DNA with tandem long terminal repeat (LTR) elements; (2) two inserts contained DNAs with deletions in or adjacent to the LTR regions; (3) a single isolate contained an inversion of 2.3 kb around the LTR in the envelope gene.     

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.     

The structure of cloned 3'-terminal RNA region of bovine leukemia virus (BLV)

cDNA synthesized on the bovine leukemia virus RNA template has been cloned in the pBR322 Pst I site. Colony hybridization with BLV RNA fragments and oligo (dT) has revealed a clone with cDNA insert containing 660 3'-terminal nucleotides of the BLV genome. The nucleotide sequence of the insert corresponding to U3 and R regions of the long terminal repeats (LTR) of viral genome has been determined. BLV U3, like U3 of other retroviruses, presumably contains promoter. The unusually long R region (about 230 bp), a certain homology with ATLV U3-R and some other structural features allow to group BLV LTR together with ATLV LTR in a separate class of retroviral LTR.     

Latency of viral expression in vivo is not related to CpG methylation in the U3 region and part of the R region of the long terminal repeat of bovine leukemia virus

Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Intramolecular integration within Moloney murine leukemia virus DNA

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features.     

Analysis of recombinant DNA clones of the endogenous BALB/c murine leukemia virus WN1802N: variation in long terminal repeat length

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.     

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.     

In vivo genomic variability of human T-cell leukemia virus type I depends more upon geography than upon pathologies.

To investigate the geography- and disease-associated genomic variation of human T-cell leukemia virus type I (HTLV-I), we studied ex vivo DNA from peripheral blood lymphocytes from nine patients by polymerase chain reaction and direct DNA sequencing. For each viral strain, 1,917 bp was sequenced, including parts of the long terminal repeat, the env gene, and the px II, px III, and px IV coding frames of the px region. The number of genomic variations observed in the U3 region of the long terminal repeat was higher than that seen in the env and px genes. Very few mutations were present in the px II and px III genes. In contrast, the px IV open reading frame exhibited numerous single point mutations. While no specific mutation could be linked to any pathology (adult T-cell leukemia/lymphoma or tropical spastic paraparesis/HTLV-I-associated myelopathy), variations among HTLV-I isolates from different geographic areas (Ivory Coast, Caribbean, and Japan) existed. The Ivory Coast HTLV-I appeared to represent a group by itself.     

Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.     

Construction and analysis of an HN-cDNA library derived from the p-arm of pig Chromosome 12

Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig × hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment. Received: 9 February 1996 / Accepted: 14 May 1996     

Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70.

We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.     

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

Structure of a defective provirus of bovine leukemia virus

Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Detection of the bovine leukaemia virus by the polymerase chain reaction.

The polymerase-chain reaction was applied for detection of provirus DNA of the bovine leukaemia virus (BLV). A short fragment of 292 bp including region R and U5 LTR 5' of BLV was amplified, and the optimum parameters of amplification of this fragment were established. Electrophoresis revealed the presence of the 292 bp fragment from the leucocytes of four out of six cows showing a positive serological response to BLV antigens. Application of the polymerase-chain reaction in diagnosis of bovine leukaemia is suggested.