The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic retrovirus.

We have recently shown that a molecularly cloned ecotropic retrovirus, initially isolated from the brain of a paralyzed wild mouse, retained the ability to induce hind limb paralysis when inoculated into susceptible mice (Jolicoeur et al., J. Virol. 45:1159-1163, 1983). To map the viral DNA sequences encoding the determinant of paralysis, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from this neurotropic murine leukemia virus (MuLV) and from nonneurotropic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered after microinjection of NIH 3T3 cells with these recombinant DNAs, were inoculated into newborn SIM.S and SWR/J mice to test the paralysis-inducing potential. We found that the 3.9-kilobase-pair SalI-ClaI fragment of the neurotropic MuLV comprising the 3' end of pol and all env sequences was sufficient to confer the paralysis-inducing potential to chimeric viruses. Therefore, this region of the neurotropic MuLV genome most likely harbors the primary determinant of paralysis.     

The envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent Friend viruses.

Friend murine leukemia virus (F-MuLV) and Friend mink cell focus-inducing virus (Fr-MCF) are helper-independent murine retroviruses which induce a rapidly fatal erytholeukemia in NIH Swiss mice. Amphotropic clone 4070 (Ampho) is a murine retrovirus which does not cause leukemia in these animals. Mice inoculated with Ampho, an Fr-MCF/Ampho pseudotype, or F-MuLV developed leukemia in 0, 50, and 100% of animals, respectively. To identify the F-MuLV and Fr-MCF sequences responsible for leukemia, we constructed hybrid viral genomes between these viruses and Ampho, using subgenomic fragments of molecularly cloned viral DNA. Transfection of these hybrid viral DNAs into fibroblasts produces recombinant retroviruses. These new viruses are assayed in vivo for their ability to cause leukemia. Recombinant viruses constructed between the Ampho genome and the Fr-MCF envelope gene do not cause leukemia. Similarly, viruses constructed by using either the Fr-MCF long terminal repeat U3 region or the F-MuLV long terminal repeat U3 region and the remainder of the Ampho genome do not cause leukemia. However, if the Fr-MCF envelope gene plus the Fr-MCF U3 region are joined to Ampho, the resulting virus causes erythroleukemia in 14% of mice. Recombinant viruses made between the Fr-MCF envelope gene, the F-MuLV U3 region, and the remainder of the Ampho genome cause erythroleukemia in 38% of mice. This study demonstrates that both the envelope gene of Fr-MCF and the U3 regions of Fr-MCF and F-MuLV contain sequences which contribute to the leukemic phenotype of helper-independent Friend viruses.     

Molecular cloning and characterization of the genomes of nine newly recognized human papillomavirus types associated with epidermodysplasia verruciformis.

The genomes of 11 human papillomaviruses (HPVs) found in benign lesions of eight patients suffering from epidermodysplasia verruciformis were cloned in Escherichia coli after insertion into plasmid pBR322. The study of the sensitivity of the cloned HPV DNAs to 14 restriction endonucleases permitted the construction of physical maps. DNA-DNA hybridization experiments, performed under stringent conditions, showed that these viruses represent nine new types, HPVs 14 (with subtypes a and b), 15, 17 (with subtypes a and b), 19, 20, 21, 22, 23, and 24. These HPVs were divided into three groups based on an absent or very weak cross-hybridization among the genomes of the viruses belonging to different groups.     

Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states

The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.     

Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis.

Five human papillomavirus (HPV) DNAs from lesions of an epidermodysplasia verruciformis patient were cloned in lambda L 47: DNA of HPV 5, which predominated in the carcinoma; DNA of a variant type of HPV 8, which was not detected in the carcinoma DNA by Southern blot hybridization but only by cloning; and DNAs of three papillomaviruses that were isolated from warts. Southern blot and liquid phase DNA-DNA hybridization under stringent conditions showed that the three viruses from warts were new types, which we named HPVs 19, 20, and 25. These viruses cross-hybridized between 3 and 29% among themselves and with HPVs 5 and 8. After physical mapping with several restriction enzymes, the colinear genomes were aligned with HPV 8 DNA to define early and late regions. HPVs 8, 19, and 25 shared homology in different parts of their genomes.     

The location of v-src in a retrovirus vector determines whether the virus is toxic or transforming.

We prepared infectious stocks of an avian retrovirus, a modified spleen necrosis virus, containing the herpes simplex virus type 1 thymidine kinase gene and the avian sarcoma virus v-src gene. Viruses were recovered after cotransfection of chicken cells with DNA of recombinants between cloned spleen necrosis virus thymidine kinase and v-src and with DNA of cloned reticuloendotheliosis virus strain A. When v-src was inserted near the 5'end of the viral genome, only low titers of recombinant virus were recovered. Most of the recovered viruses were smaller than expected and did not transform the morphology of rat or chicken cells. A very small amount of virus of the expected structure was recovered; this virus transformed rat cells and expressed v-src. Cotransfection data indicated that one reason we failed to recover a significant titer of recombinant virus is that efficient expression of v-src is acutely toxic to chicken and dog cells. Insertion of v-src near the 3' end of the viral genome, such that it was expressed at a lower level compared with the 5'-v-src-containing virus, yielded a higher titer of recombinant virus, and this virus was transforming. The differences in the recovery and transforming activity of these viruses indicate that the location of an oncogene in the viral genome is an important factor regulating the level of its expression and whether or not this expression is toxic or transforming to cells.     

Genetic individuality of intracisternal A-particles of Mus musculus.

The nucleic acid sequence relationship between mouse intracisternal type A-particles and type C and B viruses was examined by reciprocal complementary DNA-RNA hybridization; complementary DNAs prepared from the RNAs of intracisternal A-particles were hybridized with high-molecular-weight RNAs from a variety of murine tumor viruses, and complementary DNAs representing a variety of RNA tumor virus genomes were hybridized with the high-molecular-weight RNAs from A-particles. The criterion for homology between two types of virus was that the heterologous hybridization reaction occurs over the same RNA concentration range as the homologous reacton. The results of these hybridizations indicate that there are no major sequence homologies between the RNA of intracisternal A-particles and the RNA of representative members of type B and C viruses of Mus musculus.     

Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat.

The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts (E. Rassart, M. Shang, Y. Boie, and P. Jolicoeur, J. Virol. 58:96-106, 1986). To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR.     

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.     

Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively. These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12. The HPV-3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322. The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA. The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps. Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA. Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA. Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-1a, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-11a, respectively). One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

5''-terminal nucleotide sequences of mammalian type C helper viruses are conserved in the genomes of replication-defective mammalian transforming viruses.

The RNAs of replication-defective murine and primate type C transforming viruses were analyzed for the presence of nucleotide sequences homologous to the genomes of their respective helper type C viruses by using DNAs complementary (cDNA) to either the 5'-terminal (cDNA5') or total (cDNAtotal) nucleotide sequences of the helper virus RNA. The defective viruses examined have previously been shown to vary in their ability to express helper viral gag gene proteins. With cDNAtotal as a probe, these transforming viruses were shown to vary in their representation of helper sequences (15 to 60% hybridization of cDNAtotal). In striking contrast, 5'-terminal-specific sequences of the helper virus were conserved in the RNAs of every transforming virus tested (is greater than 80% hybridization of cDNA5'). These findings suggest a critical role for these sequences in the life cycle of the defective transforming virus.     

Neurotropic Cas-BR-E murine leukemia virus harbors several determinants of leukemogenicity mapping in different regions of the genome.

The infectious virus derived from the molecularly cloned genome of the neurotropic ecotropic murine Cas-BR-E retrovirus was previously shown to have retained the ability to induce hind-limb paralysis and leukemia when inoculated into susceptible mice (P. Jolicoeur, N. Nicolaiew, L. DesGroseillers, and E. Rassart, J. Virol. 45:1159-1163, 1983). To map the viral sequences encoding the leukemogenic determinant(s) of this virus, we used chimeric viral genomes constructed in vitro between cloned viral DNAs from the leukemogenic Cas-BR-E murine leukemia virus (MuLV) and from the related nonleukemogenic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered from NIH 3T3 cells microinjected with these DNAs, were inoculated into newborn NIH Swiss, SIM.S, and SWR/J mice to test their leukemogenic potential. We found that each chimeric MuLV, harboring either the long terminal repeat, the gag-pol, or the pol-env region of the Cas-BR-E MuLV genome, was leukemogenic, indicating that this virus harbors several determinants of leukemogenicity mapping in different regions of its genome. This result suggests that the amphotropic 4070-A MuLV has multiple regions along its genome which prevent the expression of its leukemogenic phenotype, and it also shows that substitution of only one of these regions for Cas-BR-E MuLV sequences is sufficient to make it leukemogenic.     

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.     

Genesis of Kirsten murine sarcoma virus: sequence analysis reveals recombination points and potential leukaemogenic determinant on parental leukaemia virus genome.

The genome of Kirsten murine sarcoma virus was formed by recombination between Kirsten murine leukaemia virus sequences, and rat sequences derived from a retrovirus-like '30S' (VL30) genetic element encompassing the Kras oncogene. Using cloned DNAs we have determined the nucleotide sequences of the long terminal repeats and adjacent regions, extending across the points of recombination on the sarcoma and leukaemia virus genomes. Our results suggest that discrete regions of homology and other cryptic sequence features, may have constituted recombinational hot-spots involved in the genesis of the Kirsten murine sarcoma virus genome. We have also compared the sequence of the Kirsten murine leukaemia virus p15 env and adjacent long terminal repeat with the corresponding regions of the AKV and Gross A murine leukaemia virus genomes. This comparison has identified a leukaemogenic determinant in the U3 domain of the long terminal repeat, possibly within a enhancer-like sequence element.     

Nucleic Acid Homology of Murine Type-C Viral Genes

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [³H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies Mus musculus molossinus and M. musculus castaneus. All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.     

Free and integrated recombinant murine leukemia virus DNAs appear in preleukemic thymuses of AKR/J mice.

We studied the appearance and structure of murine leukemia viral genomes in preleukemic AKR/J mice by Southern hybridization. Up to an average of one to two copies per thymocyte of unintegrated murine leukemia virus DNA appears in the thymuses of preleukemic mice beginning at 4 to 5 months of age and disappears in leukemic thymuses. The free viral genomes are absent in the spleens, livers, and brains of preleukemic mice. Using a series of ecotropic and nonecotropic murine leukemia virus hybridization probes, we showed that the unintegrated viral genomes are structurally analogous to those of recombinant mink cell focus-forming viruses that appear as proviruses in leukemic AKR thymocytes, suggesting that these free viral DNAs are the direct precursors to the leukemia-specific proviruses. The mosaic of ecotropic and nonecotropic sequences within these unintegrated viral DNAs varies from one preleukemic thymus to another but often appears structurally homogeneous within individual thymuses, indicating that often each thymus was being infected by a unique mink cell focus-forming virus. Analysis of high-molecular-weight DNA shows that recombinant proviruses reside in the chromosomal DNA of thymocytes within the preleukemic thymus, with the number rising to an average of several copies per thymocyte, but we do not detect any preferred integration sites. These results suggest that, in general, before the development of thymic leukemias in AKR mice there is a massive infection by a unique mink cell focus-forming virus which then integrates into many different sites of individual thymocytes, one of which grows out to become a tumor.     

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection.The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.