Infectious Viral DNA Research Articles

Important role of the long terminal repeat of the helper Moloney murine leukemia virus in Abelson virus-induced lymphoma.

The helper virus has been shown to play a critical role in the development of lymphoma induced by the defective Abelson murine leukemia virus (A-MuLV). Indeed, A-MuLV pseudotyped with some viruses, such as the Moloney MuLV, has been shown to be highly lymphogenic, whereas A-MuLV pseudotyped with other viruses, such as the BALB/c endogenous N-tropic MuLV, has been shown to be devoid of lymphogenic potential (N. Rosenberg and D. Baltimore, J. Exp. Med. 147:1126-1141, 1978; C. D. Scher, J. Exp. Med. 147: 1044-1053, 1978). To map the viral DNA sequences encoding the determinant of the lymphogenic potential of Moloney MuLV when complexed with A-MuLV, we constructed chimeric helper viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from Moloney MuLV and from BALB/c endogenous N-tropic MuLV. Chimeric helper MuLVs, recovered after transfection of NIH 3T3 cells were used to rescue A-MuLV, and the pseudotypes were inoculated into newborn NIH Swiss, CD-1, and SWR/J mice to test their lymphogenic potential. We found that a 0.44-kilobase-pair PstI-KpnI long terminal repeat-containing fragment from the Moloney MuLV was sufficient to confer some, but not complete, lymphogenic potential to a chimeric virus (p7M2) in NIH Swiss and SWR/J mice, but not in CD-1 mice. The addition of the 3'-end env sequences (comprising the carboxy terminus of gp70 and all p15E) to the U3 long terminal repeat sequences restored the full lymphogenic potential of the Moloney MuLV. Our data indicate that the 3'-end sequences of the helper Moloney MuLV are somehow involved in the development of lymphoma induced by A-MuLV. The same sequences have previously been found to harbor the determinant of leukemogenicity and of disease specificity of Moloney MuLV when inoculated alone.

Resolution of a polyomavirus-mouse hybrid replicon: release of genomic viral DNA.

RmI is a circular chimera containing 1.03 copies of polyomavirus DNA and 1,628 base pairs of mouse DNA, joined through direct and inverted repeat sequences. It is excised from the chromosome of a transformed cell via a site-specific recombination event that is dependent on the activation of the viral gene coding for large T antigen. RmI is shown here to be highly infectious for normal mouse cells. This infectivity reflects the ability of RmI to effectively yield unit-length viral DNA via intramolecular recombination. The effectiveness with which infectious viral DNA is produced from RmI is consistent with the idea that the underlying recombination event is site specific, rather than homologous or illegitimate.

“Agroinfection,” an alternative route for viral infection of plants by using the Ti plasmid

Most plant viruses are transmitted by insect vectors. We present an alternative method for the introduction of infectious viral DNA that uses the ability of Agrobacterium to transfer DNA from bacterial cells to plants. Cauliflower mosaic virus was chosen to develop this method because it is the best characterized plant DNA virus and can be introduced into plants via aphids, virus particles, viral DNA, or suitably treated cloned DNA. We show that systemic infection of turnips results from wounding and inoculation with strains of Agrobacterium tumefaciens in which more than one genome of cauliflower mosaic virus have been placed tandemly in the T-DNA of the tumor-inducing plasmid. Thus such constructions allow escape of the viral genome from the T-DNA once inside the plants. The combined use of the tumor-inducing plasmid and viral DNA opens the way to molecular biological approaches that are not possible with either system alone.

Recombination in a plant virus: template-switching in cauliflower mosaic virus

A hybrid plasmid, containing tandemly arranged pieces of two different but well-defined cauliflower mosaic virus (CaMV) genomes, was used to study the mechanism by which infectious viral DNA can escape from transforming DNA. Systemic viral infection followed inoculation of Brassica plants with a strain of Agrobacterium tumefaciens containing the hybrid plasmid in its T-DNA. Restriction mapping of uncloned viral DNA from these plants, and sequencing of relevant portions of cloned viral DNA, showed that the majority of viral progeny were probably descendants of DNA produced by transcription/reverse transcription of the viral genome, thus providing further evidence for the hypothesis that this process is normally involved in viral replication. The reverse transcriptase enzyme, which is thought to undergo an intramolecular template switch during viral replication, is shown to move very close to the 5' end of the terminal repeat on the 35S RNA molecule before switching templates. The remaining minority of viral genomes can best be explained as arising from products of recombination between homologous regions of CaMV DNA.

Mapping the viral sequences conferring leukemogenicity and disease specificity in Moloney and amphotropic murine leukemia viruses.

The Moloney murine leukemia virus (MuLV) is a highly leukemogenic virus. To map the leukemogenic potential of Moloney MuLV, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA from Moloney and amphotropic 4070-A MuLVs. Infectious chimeric MuLVs were recovered by microinjection of recombinant DNA into NIH/3T3 cells and tested for their leukemogenic potential by inoculation into NIH/Swiss newborn mice. Parental Moloney MuLV and amphotropic 4070-A MuLV induced thymic and nonthymic leukemia, respectively, when inoculated intrathymically. With chimeric MuLVs, we found that the primary determinant of leukemogenicity of Moloney and amphotropic MuLVs lies within the 1.5-kilobase-pair ClaI-PvuI long terminal repeat (LTR)-containing fragment. The presence of additional Moloney env-pol sequences with the Moloney LTR enhanced the leukemogenic potential of a chimeric MuLV significantly, indicating that these sequences were also involved in tumor development. Since parental viruses induced different forms of leukemia, we could also map the viral sequences conferring this disease specificity. We found that the 1.5-kilobase-pair ClaI-PvuI LTR-containing fragment of Moloney MuLV was necessary and sufficient for a chimeric MuLV to induce thymic leukemia. Similarly, the same LTR-containing fragment of amphotropic MuLV was necessary and sufficient for a chimeric MuLV to induce nonthymic leukemia. Therefore, our results suggest that specific sequences within this short LTR-containing fragment determine two important viral functions: the ability to transform cells in vivo (leukemic transformation) and the selection of a specific population of cells to be transformed (disease specificity).

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.

Fv-1 host cell restriction of friend leukemia virus: microinjection of unintegrated viral DNA

The murine gene Fv-1 has been shown to exert a major influence over the replication of ecotropic murine leukemia viruses. Studies of the replication of Friend murine leukemia virus have shown that the restriction of viral replication occurs intracellularly after the initiation of viral DNA synthesis. The precise mechanism of the block imposed by the Fv-1 gene product is not completely understood. Our studies of Fv-1 restrictive infection have shown a variable decrease in the accumulation of intracellular unintegrated form I viral DNA. Analysis by microinjection of the viral DNA formed in nonpermissively infected BALB/c cells indicates that this DNA is infectious. These studies indicate that the form I DNA accumulated in nonpermissively infected BALB/c cells contains the complete viral sequences necessary for the production of viral progeny, and therefore, they suggest that the Fv-1 host restrictive mechanism recognizes viral factors other than form I DNA alone. These results support the possibility that Fv-1 host restriction occurs after formation of infectious viral DNA, perhaps at the integration step itself.

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.

Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus.

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.

Complementation of a tsa mutant and replication of a recombinant DNA carrying the viral ori region in mouse cells transformed by polyoma virus

Two independent procedures are described that yield polyoma virus-transformed mouse cell lines expressing viral large-T protein but unable to synthesize either infectious virus or free viral DNA. The first procedure uses a recombinant plasmid carrying the promotor- ori region of SV40 virus linked to the early coding region of polyoma. In the second procedure, transformants are isolated after transfer of an entire polyoma genome linked to bacterial plasmid pBR322 sequences. These procedures can be applied either to normal mouse cells, using selection for the transformed growth phenotype (focus formation, colony formation in agarose medium), or to already transformed cells, by cotransfer of a selectable genetic marker. These polyoma transformant mouse cells remain permissive for virus growth and keep their ability to synthesize a functional large-T protein, even upon serial transfers in culture. Like the previously described cos cells in the case of SV40 virus, the “ scop” lines can complement an early is mutant for growth at high temperature and replicate a recombinant DNA molecule that includes the origin of replication of polyoma DNA.

Elektronenoptische Untersuchungen zur in vitro‐Wirkung von Dipyridamol auf das Pseudorabiesvirus

AbstractDipyridamole at a concentration of 50 μM/ml displays no activity on adsorption and penetration of pseudorabies virus in chicken embryonal cells. Furthermore, first stages of virus replication take place within the nucleus, whereas incomplete virus cores defective in DNA content are found within the nucleoplasm at times when the regular viral replication has been finished in controls. Defective pseudorabies virus particles lacking in DNA‐content of the core, can be observed at the end of normal replication time. Consequently, the antiviral activity of dipyridamole may be due to blocking of the synthesis or of the incoporation of infectious viral DNA into the virus core.

Electron microscopic study of the in vitro effect of dipyridamol on the pseudorabies virus

Dipyridamole at a concentration of 50 microM/ml displays no activity on adsorption and penetration of pseudorabies virus in chicken embryonal cells. Furthermore, first stages of virus replication take place within the nucleus, whereas incomplete virus cores defective in DNA content are found within the nucleoplasm at times when the regular viral replication has been finished in controls. Defective pseudorabies virus particles lacking in DNA-content of the core, can be observed at the end of normal replication time. Consequently, the antiviral activity of dipyridamole may be due to blocking of the synthesis or of the incorporation of infectious viral DNA into the virus core.

Nucleotide sequence of the genome of a murine sarcoma virus

The complete nucleotide sequence of a molecularly cloned, biologically infectious mouse sarcoma viral DNA has been determined. It is 5,833 nucleotides in length. It contains two long terminal repeats, the coding capacity for the entire gag gene, portions of the pol gene and the entire transforming gene. Synthesis of the 538 amino acid-long gag gene-encoded polyprotein Pr62 gag initiates 596 nucleotides downstream from the 5′-cap nucleotide. The cleavage of gag polyprotein to mature core proteins, NH 2-p15-p12-p30-p10-COOH, does not involve the loss of any amino acids. The complete nucleotide sequence of the cellular homolog (c- mos Mo) of viral transforming gene v- mos Mo has been determined. There are 25 single-base changes in an uninterrupted sequence of 1,157 nucleotides. Of these, 21 changes are in the coding region and they alter only 11 amino acids with respect to v- mos Mo gene product. The homologous and nonhomologous regions between mouse sarcoma viral DNA and its progenitor, murine leukemia viral DNA, have been identified. Most deletions appear to have occurred by homologous recombination. A sequence GACC or its inverse GGTC is present in the vicinity of all substitutions and deletions. The codon usage of the gag gene is quite different from that of the c- mos Mo gene.

Recovery of biologically active spleen focus-forming virus from molecularly cloned spleen focus-forming virus-pBR322 circular DNA by cotransfection with infectious type C retroviral DNA.

The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.

Molecular Cloning of Polyoma Virus DNA in Escherichia coli : Plasmid Vector System

A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells. While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection.

The Kinetics of Synthesis of Infectious Unintegrated Virus DNA in Rous Sarcoma Virus Infected Chicken Cells

Infectious virus nucleic acids were extracted from the Hirt supernatant of chicken embryo fibroblasts infected for different times with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). Infectious DNA and DNA-RNA hybrid molecules could be recovered from 6 h after infection in experiments using 1.5 X 10(9) infected cells. Only small amounts of infectious virus DNA could be purified 6 h after infection whereas at 24 h approximately one infectious DNA molecule could be recovered for each input virus infectious unit. At 24 h, both infectious supercoiled and non-supercoiled molecules were found. The specific infectivity of the supercoiled fraction was less than that of the non-supercoiled fraction. Infectious supercoiled DNA could be recovered from 16 h after infection. Evidence is presented that both forms of unintegrated virus DNA may rest unintegrated for at least 8 days in the cell, though chronically infected cells were shown to contain less than one unintegrated molecule per 10(2) to 10(3) cells.

In vitro Synthesis of Infectious DNA Copies of the Rous Sarcoma Virus Genome

Detergent-disrupted virions of Rous sarcoma virus can synthesize in vitro an infectious viral DNA. Infectivity of the DNA was determined in transfection assays at the endpoint dilution. It was found that the specific infectivity of the crude endogenous reaction product was 34 infectious units/microgram. The endogenous reaction yielded up to 8.5 x 10(-7) infectious units per biologically active input virion, i.e., nearly 0.1 -1 infectious molecules. The possible structure of these molecules is discussed.

The search for infectious viral DNA in human leukemic cells.

That human leukemia has a viral origin has been suggested many times (see [6] for references). Leukemic cells have been shown to bear intracellular virus-like particles [1,4], to share nucleotide sequences with murine [1] or primate [4] viruses, and to synthesize proteins that cross-react with antibodies to virus-specific proteins [18]. Some authors reported the release of C-type particles from leukemic cells subjected to in vitro culture conditions [11,12,13,16], and Gallagher and Gallo [3] have shown budding C-type virus in leukemic cells sustained proliferating in vitro with medium conditioned by a particular human embryonic cell culture. This virus, now called HL23V, was further studied and found to be infectious for a wide variety of cells. Viruses growing in these new hosts were referred to as “secondary viruses” [20].KeywordsHuman Leukemic CellTransfection AssayReverse Transcriptase ActivityAvian Leukosis VirusChicken CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Induction of Infectious Virus DNA and Virus Particles by Mitomycin C in SV40-Transformed Mouse Cells

A line of SV40-transformed mouse cells (SV/3T3-4E) was isolated and four clones were derived from this line. Spontaneous production of small amounts of infectious SV40 DNA was detected in the parental line and in three out of the four clones tested, although no synthesis of virions could be demonstrated. The yields of SV40 DNA were significantly enhanced following treatment of these cells with mitomycin C and infectious virus particles became detectable occasionally.

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective ( td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10 −1 to 10 −4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.