Search PubMed⌕ Search

Biomedical subjects

H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 235 records · Page 13Linked to original sources
PubMed1977

Oncogenicity of avian leukosis viruses of different subgroups and of mutants of sarcoma viruses.

Leukosis viruses of seven subgroups were tested for oncogenicity in chickens susceptible to virus infection and to development of lymphoid leukosis (LL) tumors. All subgroup A viruses and the subgroup B virus tested produced a high incidence of LL and other related neoplasms. Viruses of subgroup C and RAV-61 of subgroup F produced a low level of LL. The RAV-50 of subgroup D produced osteopetrosis. In these tests, the viruses of subgroup E and G and one virus of subgroup F were not pathogenic, possibly because infection was not established in the chickens, the chickens were not susceptible to tumor development by these viruses, or the viruses lacked oncogenicity. All temperature-sensitive mutants of Rous sarcoma virus produced sarcomas, but the level varied. One nontransforming mutant produced sarcomas, and the other three tested produced LL. All three mutants that cause cells to grow as colonies in agar produced a high incidence of sarcomas. Thus, sarcoma viruses, by back-mutation, may lose the ability to transform cells in vitro, to make cells grow in agar colonies, or to induce sarcomas in vivo, yet they retain the ability to produce LL. Conversely, it was previously shown that leukosis viruses may be changed into viruses that transform cells in vitro and produce sarcomas in vivo by suitable passage in chicks.

Animals↗
PubMed1977

Formation of reticuloendotheliosis virus pseudotypes of Rous sarcoma virus.

Superinfection of chicken embryo fibroblasts transformed by the defective Bryan strain of Rous sarcoma virus (BH-RSV) with two different reticuloendotheliosis viruses (REVs), REV strain T (REV-T) or spleen necrosis virus (SNV), resulted in the production of infectious sarcoma virus pseudotypes. These pseudotypes were neutralized by antiserum prepared against SNV and were unable to infect chicken cells preinfected with either REV-T or SNV. These results suggest that defective BH-RSV is able to use the glycoprotein from REV to form infectious pseudotypes. On the other hand, neither REV-T nor SNV was able to supply a functional reverse transcriptase to the polymerase-negative mutant BH-RSValpha, nor was REV-T or SNV able to complement the defect in the internal protein gene of the temperature-sensitive avian sarcoma virus mutant NY45.

Animals↗
PubMed1977

Genetic variation in the RNA transcripts of endogenous virus genes in uninfected chicken cells.

Uninfected cells from two different phenotypes of chicken embryos express significant amounts of endogenous viral information, though they do not produce virus particles. Cells of the phenotype gs(+)chf(+) are positive for both group-specific (gs) antigens and chicken helper factor (chf) activity, whereas cells of a second phenotype, gs(L)chf(+)(h(E)), demonstrate noncoordinate expression of these two viral activities (very low amounts of gs antigens, but extremely high helper activity). RNA from these cells was analyzed to determine the size, genetic content, and relative abundance of virus-specific RNAs in cells of each phenotype. Two major size classes of polyadenylic acid-containing RNA, homologous to the avian leukosis virus genome, were detectable in cells of both types. The larger RNA, which contained most of the sequences of the leukosis virus genome, was of different sizes in the two phenotypes, 31S in gs(+)chf(+) cells but 35S in the noncoordinate cell type. Analysis of the viral RNA with gene-specific complementary DNA probes revealed the following characteristics. (i) The 31S RNA appeared to lack portions of the gag and pol genes. (ii) A smaller RNA species, which sedimented at 21S in both cell types, was a transcript of the 3'-proximal portion of the viral genome, consisting of the env gene and the "common" sequences. (iii) The amount of env-specific RNA in the 21S region was more than six times higher in the noncoordinate cell type than in the gs(+)chf(+) cells; this difference was concordant with the 5- to 10-fold higher chf activity in the noncoordinate cells. (iv) The endogenous viral RNA in uninfected cells and the RNA from Rous-associated virus-0 virions hybridized only partially with DNA complementary to the common region of the Rous-associated virus-2 genome, whereas the RNA of all exogenous viruses tested hybridized almost completely to this complementary DNA. Small amounts of src-specific polyadenylated RNA were also present in uninfected chicken cells. This RNA sedimented as a single peak at 26S and was not covalently linked to any other identifiable virus-specific RNA sequences. The amount of src RNA was the same in the above two types of expression-positive cells and also in cells that were gs(-)chf(-), indicating that the transcription of the cellular sequences homologous to the src gene is independent of the transcription of the other endogenous viral genes.

Animals↗
PubMed1977

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.

Autoradiography↗
PubMed1977

Transformation-defective mutants of Rous sarcoma virus with src gene deletions of varying length.

The RNAs of transformation-defective (td) deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus were found to vary in size when compared by polyacrylamide gel electrophoresis. Three of seven td mutants appeared to recombine with a mutant of Rous sarcoma virus (Schmidt-Ruppin), which has a temperature-sensitive sarcoma (src) gene and is termed ts68, to give rise to recombinants with a reduced temperature sensitivity. The results suggested that different clones of td mutants exist: some in which the src gene appears to be deleted, and others in which the src gene is only partially deleted. A direct correlation between RNA size and the extent of src gene deletion measured by recombination was not obtained, possibly because the recombination assay could only detect src sequences homologous to the lesion(s) of ts68, whereas the electrophoretic analysis of the RNA measured src deletions as well as other possible alterations of the RNA.

Avian Sarcoma Viruses↗
PubMed1976

Sequences and functions of Rous sarcoma virus RNA.

A procedure has been developed to map the genetic elements of avian tumor virus RNA, which has a molecular weight of about 3 X 10(6) daltons and a poly(A) sequence at the 3' end. For this purpose, about 30 RNase T1-resistant oligonucleotides were ordered relative to the 3'-poly(A) terminus of the RNA, to construct an oligonucleotide map of viral RNAs. A cluster of seven envelope gene (env)-specific oligonucleotides, identified by their absence from the otherwise very similar oligonucleotide map of an envelope-defective deletion mutant (which lacks the major viral glycoprotein), mapped at a distance of 0.9 to 1.6 X 10(6) daltons from the poly(A) end of sarcoma virus RNA. A cluster of three sarcoma gene (src)-specific oligonucleotides, identified by their absence from the otherwise nearly identical oligonucleotide map of a transformation-defective deletion mutant mapped at a distance of 0.2 to 0.6 X 10(6) daltons from the poly(A) end of sarcoma virus RNA. The oligonucleotide maps of sarcoma viruses and of related deletion mutants were the same from the poly(A) end up to 0.2 X 10(6) daltons and included one terminal oligonucleotide, termed C, which is found in all avian tumor viruses tested so far. Preliminary mapping experiments ordering the src-specific and env-specific oligonucleotides of recombinants, selected for sarcoma and envelope genes of different parents, agree with those obtained by comparing maps of wild type viruses and deletion mutants. A partial genetic map consistent with these results suggests that the src gene maps between the env gene and the 3'-poly(A) end of viral RNA. This map reads: poly(A)-src-env-(pol, gag).

Avian Sarcoma Viruses↗
PubMed1976

Location of envelope-specific and sarcoma-specific oligonucleotides on RNA of Schmidt-Ruppin Rous sarcoma virus.

Envelope-specific and sarcoma-specific nucleotide sequences have been located within the 10,000 nucleotides of the RNA of nondefective Schmidt-Ruppin Rous sarcoma virus (nd SR). For this purpose, about 30 RNase-T1-resistant oligonucleotides were ordered relative to the 3'-poly(A) terminus of the RNA, to construct an oligonucleotide map of the nd SR RNA. A cluster of seven envelope-specific oligonucleotides, identified by their absence from an otherwise very similar oligonucleotide map of an envelop-defective deletion mutant (which lacks the major viral glycoprotein), mapped at a distance of 2800-5000 nucleotides from the poly(A) end of nd SR RNA. A cluster of two sarcoma-specific oligonucleotides, identified by their absence from an otherwise nearly identical oligonucleotide map of a transformation-defective deletion mutant, mapped at a distance of 1000-2000 nucleotides from the poly(A) end of nd SR RNA. The oligonucleotide maps of nd SR and of the two deletion mutants were the same from the poly(A) end up to 650 nucleotides and included one terminal oligonucleotid, termed C, which is found in all avian tumor viruses tested so far. A possible gene order consistent with our data suggests that sarcoma-specific nucleotide sequences map between envelope-specific nucleotide sequences and the poly(A) end of the RNA.

Avian Sarcoma Viruses↗
PubMed1976

Pheasant virus: new class of ribodeoxyvirus.

Cocultivation of cells derived from embryos of golden pheasants or Amherst pheasants with chicken embryo cells infected with Bryan strain of Rous sarcoma virus resulted in the detection of viruses which appear to be endogenous in these pheasant cells. The pheasant viruses (PV) were similar to avian leukosis-sarcoma viruses (ALSV) in their gross morphology, in the size of their RNA, in the presence of a virion-associated RNA-dependent DNA polymerase (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7), and in their growth characteristics. PV also serves as a helper for the glycoprotein-defective Rous sarcoma virus. However, PV was shown to be different from both ALSV and reticuloendotheliosis virus in the following properties: (i) PV does not have ALSV group specific antigens; (ii) the protein composition of PV is different from those of the other two groups of viruses; (iii) PV fails to complement the defective polymerase of alpha type Rous sarcoma virus; and (iv) PV RNA shows no detectable homology with nucleic acids of the other two groups of viruses. Thus, PV appears to be a new class of RNA viruses which contain RNA-dependent DNA polymerase.

Alpharetrovirus↗
PubMed1976

Independent regulation of endogenous and exogenous avian RNA tumor virus genes.

3H-Labeled complementary DNA specific for the envelope glycoprotein (env) gene of avian leukosis-sarcoma viruses was isolated by selective nucleic acid hybridization techniques, and used to analyze the expression of the endogenous provirus. The endogenous provirus in certain cell types termed chicken helper factor positive (chf+) can synthesize the envelope glycoprotein. Env DNA sequences were present in both chf+ and chf- cells, but env RNA was detectable only in positive cell types. When these cells were infected with the Bryan strain of Rous sarcoma virus (BH-RSV), a defective virus which is deleted in the env gene, the levels of endogenous env RNA remained unchanged, although exogenous BH-RSV specific RNA was synthesized in very high amounts. Thus, the infecting virus did not appear to influence the expression of the endogenous virus. Likewise, the endogenous virus did not influence the exogenous virus expression, since similar amounts of BH-RSV specific RNA were present in all infected cell types, regardless of the level of endogenous virus expression.

Alpharetrovirus↗
PubMed1976

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.

Animals↗
PubMed1976

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.

Animals↗
PubMed1975

RNA of replication-defective strains of Rous sarcoma virus.

The RNA of a replication-defective (rd) mutant, isolated from stocks of nondefective (nd) Schmidt-Ruppin Rous sarcoma virus of subgroup A (SR-A) and termed SR-N8, was compared to the RNAs of SR-A, of a transformation-defective derivative of SR-A (td SR-A) and of rd Bryan Rous sarcoma virus, RSV (minus). The molecular mass of the 30-40S species of SR-N8 RNA was estimated to be 21% (congruent to 7.5 to 8 times 10-5 daltons) smaller than that of SR-A by (i) electrophoresis in polyacrylamide gels and (ii) analyses of RNA complexity based on RNase T1-resistant oligonucleotides. ST-N8 shares probably all (=14) of its large RNase T1-resistant oligonucleotides with the RNA of SR-A as judged from the chromatographic distribution and the RNase A-resistant fragments obtained from RNase T1-resistant oligonucleotides. However, SR-N8 RNA lacked six large oligonucleotides which were present in the RNAs of SR-A and td SR-A. Conversely, the RNAs of SR-A, and of SR-N8 contained two oligonucleotides not found in td SR-A. The RNA of SR-N8 was found to differ from that of RSV (minus) in its electrophoretic mobility and its fingerprint pattern. It is concluded that the RNA of SR-N8 was generated by a deletion of SR-A. The extent of this deletion is compatible with the notion that the genetic information for the large viral envelope glycoprotein (molecular mass = 70,000-85,000 daltons) has been lost from the RNA of SR-A to yield SR-N8 RNA. From a comparison of td and rd deletion mutants, it appears that loss of different functions corresponds to the absence of different oligonucleotides in their RNA.

Avian Sarcoma Viruses↗
PubMed1975

Relationship between A-type and C-type particles in cells infected by Rous sarcoma virus.

Chicken cells infected with avian RNA tumor virus often contain small cytoplasmic A-type particles which commonly exist as clusters of 50--100 particles when viewed in thin sections. These particles were found more consistently in Rous sarcoma virus-infected than Rous-associated virus-infected cultures, but were generally present in only a small fraction of the total infected cells. The results of the survey of cells infected with various strains of leukosis-sarcoma viruses led to the hypothesis that the A particles develop in cells undergoing cytopathological degeneration. The hypothesis explains also the evanescent nature of the appearance of these particles in infected cells. The application of immunoelectron microscopic methods using monospecific antisera against viral internal proteins revealed that the A particles contain components immunologically related to the proteins of C-type virus.

Animals↗
PubMed1975

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.

Animals↗