Prague Strain Of Rous Sarcoma Virus Research Articles

Remembering Jan Svoboda: A Personal Reflection

The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term persistence of the viral genome in non-productive mammalian cells, and he supported the DNA provirus hypothesis of Howard Temin. He showed how the virus can be rescued in the infectious form and elucidated the replication-competent nature of the Prague strain of RSV later used for the identification of the src oncogene. His studies straddled molecular oncology and virology, and he remained an active contributor to the field until his death last year. Throughout the 50 years that I was privileged to know Svoboda as my mentor and friend, I admired his depth of scientific inquiry and his steadfast integrity in the face of political oppression.

Ducks: a new experimental host system for studying persistent infection with avian leukaemia retroviruses

Long-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.

Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site.

Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed.

Truncated gag-related proteins are produced by large deletion mutants of Rous sarcoma virus and form virus particles.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, contain two deletions relative to wild-type virus. One of these joins gag sequences in the p12 coding region to env sequences in region encoding gp37; the other deletion spans the src region. Analysis of the viral proteins of QT6 cell clones containing only LD proviruses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major truncated gag-related phosphoprotein of 60,000 to 66,000 daltons (P63LD). P63LD was stable, but could be cleaved in vitro to the predicted products by p15gag. A second gag-related LD protein of about 68,000 to 74,000 molecular weight (P70LD) was also found which often reacted with an anti-gp37 serum. P70LD was unstable and may represent a short-lived gag-gp37 fusion protein. Finally, immunoprecipitation indicated that particles containing P63LD were shed from QT6-LD clones. Thin section preparations of these clones viewed in an electron microscope showed enveloped budding particles of "immature" morphology. Thus, the synthesis and release of particles from infected cells does not require cleavage of the gag precursor, nor does it require the presence of p15 or (most of) p12.

Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus.

The 3' terminal region of the Prague strain of Rous sarcoma virus (PrRSV) contains at least three distinct domains that comprise two functional enhancer elements. Two of these domains (designated B and C) are found in the U3 region of the 3' long terminal repeat (LTR) while the third (designated A) is located in the sequences immediately preceding the LTR termed XSR sequences. Combinations of adjacent domains [e.g., (A + B or B + C)] are capable of activating the expression of the SV40 early promoter (21 bp repeats and TATA box) coupled to coding sequences from the prokaryotic gene chloramphenicol acetyltransferase (CAT) while a single domain is inactive. Furthermore, duplication or triplication of the central domain B restores activity. The related, Schmidt-Ruppin, strain of RSV, contains an almost identical 3' LTR element, but differs in the enhancer sequences immediately preceding the 3' LTR. A model is presented in which the sequence differences may contribute to the difference in disease spectrum of transformation defective (td) variants of these viruses.

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Restriction endonuclease EcoRI digestion of the viral DNA of 12 nonconditional transformation defective ( td) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an EcoRI B ( src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase EcoRI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src (Czernilovsky et al., Nature (London) 287, 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src, and in some cases also in regions between src and env, leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive ( ts) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src, 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src. A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology ( td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.

Novel nonconditional mutants in the src gene of rous sarcoma virus: Isolation and preliminary characterization

We have isolated a number of nonconditional transformation-defective ( td) mutants of Prague strain Rous sarcoma virus, subgroup A (PR-RSV-A). Many of these resembled td mutants reported previously, but 11 isolates from low-passage stocks of PR--A showed unusual properties and were designated partially td (ptd) mutants. In mixed infections with temperature-sensitive ( ts) transformation-defective RSV mutants the ptd viruses produced cell transformation at restrictive temperature (41°), probably by genetic recombination to yield wild-type virus. In tests with a panel of 4 ts mutants, we found that different ptd isolates varied in the number and pattern of ts mutants with which they showed this effect. In mixed infections with one another the ptd viruses yielded transforming virus. Again, the pattern shown by different ptd viruses varied, and on the basis of this variation the 11 ptd isolates appear to comprise at least 10 distinct mutants. The possibility of genome deletions in some of the viruses was examined in Southern blots of EcoRI digests of proviral DNA. Two ptd viruses, which recombined with all 4 ts mutants tested, had EcoRI restriction fragments identical to those of wild-type PR-A. Three isolates which recombined with either 3, 2, or none of the ts mutants, showed deletions in the EcoRI fragment containing the src gene. These deletions corresponded to losses of 1.0, 1.5, and 1.6 kilobases, respectively, from the RNA genome. We conclude that these ptd viruses bear either point mutations or deletions of varying size but all retain part of the src gene. These mutants are stable and should be useful for further genetic and physiological studies on the src gene and its product.

The Influence of Host Adaptation of Rous Sarcoma Virus on the Transfecting Activity of its DNA Provirus

Mammalian cells transformed with either Prague strain Rous sarcoma virus of subgroup C (XC cells) or Schmidt-Ruppin strain Rous sarcoma virus of subgroup D (RSCH cells) yielded virus upon fusion with chick cells. Virus was also rescued by transfection of DNA from these cells on to chick cells. However, virus rescue did not occur upon transfection of duck cells, and fusion with duck cells led to virus rescue only from RSCH and not from XC cells. To investigate this restriction on the duck cells the non-defective Prague strain of Rous sarcoma virus of subgroup C (PR-RSV-C) was adapted for efficient replication in duck embryo cells (daPR-RSV-C) by long-term passage in vitro. However, a second PR-RSV-C isolate, rescued from the rat XC sarcoma line (XC DNA 940 virus), failed to adapt to growth in duck cells. When transformed with daPR-RSV-C, which replicates in duck cells as well as in brown leghorn embryo (BLEF) cells, duck cells yielded DNA which transfects fresh duck cells, in contrast to DNA isolated from chicken or duck cells transformed with parental PR-RSV-C.

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.

Src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60(src) from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avain sarcoma virus (ASV)], the molecular weights (M(r)s) ranging from 60,000 to 64,000. The p60(src) immunoprecipitated from cells transformed by each of these strains incorporated [gamma-(32)P]ATP into the M(r) 53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent K(m) for ATP of 4.9 +/- 2 muM, and was dependent on Mg(2+) (K(b) = 46 +/- 12 mM), for which Ca(2+) was no substitute. The kinase was cyclic AMP independent. In order to test whether the protein kinase reaction is directly catalyzed by p60(src), we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60(src). Concomitant with the loss of the kinase activity by heat inactivation, p60(src) loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60(src) kinase is itself dependent on phosphorylation for its activity.

Nucleotide sequence relationships between the genomes of an endogenous and an exogenous avian tumor virus

We have used mapping of large T1 oligonucleotides to examine the genome of Rous-associated virus-O (RAV-O), an endogenous virus of chickens, and to compare it with that of Prague strain Rous sarcoma virus, subgroup B, (Pr-RSV-B), an exogenous sarcoma virus. To extend the sensitivity of such comparisons, we have developed a system of nucleic acid hybridization and hybridization-competition combined with fingerprinting. This method allows us to estimate the relative degree of relatedness of various portions of the viral genomes. From the results of this study, we have concluded that the genomes of Pr-RSV-B and RAV-O are related in the following way. The 5'-terminal half of the genomes (corresponding to the gag and pol regions) is virtually identical, with only scattered single nucleotide differences. This region is followed by a region comprising 25 to 30% of the genome (the env region) which contains substantial nucleotide sequence differences, most or all of which are due to single base changes. The env-coding region can be further subdivided into three regions: a more variable region probably containing sequences coding for subgroup specificity, flanked by relatively common sequences on each side. To the 3' side of the env region, the RAV-O genome contains a very short sequence not found in Pr-RSV-B, whereas the Pr-RSV-B genome contains a much longer unrelated sequence. The central portion of this sequence comprises the src gene as defined by transformation-defective mutants. Particularly striking is the absence, in the RAV-O genome, of any nucleotide sequence related to the "c region" found very near the 3' end of all exogenous tumor viruses. Both the Pr-RSV-B and RAV-O genomes contain the identical terminally redundant sequence of 21 nucleotides near each end of the genome.

A mutant of rous sarcoma virus with a thermolabile defect in the virus envelope

A mutant of Prague strain Rous sarcoma virus, ts LA30m PR-A (referred to here as LA30A), has a temperature-sensitive ( ts) defect early in infection. The defect appears to reside in a structural component of the virus, for mutant virions are heat labile. Two observations show that the mutated virion components function very early during infection. First, LA30A passes through the ts stage of its life cycle more rapidly than another early mutant which has a ts RNA-dependent DNA polymerase, indicating that the defective LA30A function operates before reverse transcription. Second, once LA30A has penetrated the cell, the infection is no longer ts. Pseudotypes of both Bryan high-titer strain Rous sarcoma virus and of vesicular stomatitis virus with envelopes donated by LA30A show heat lability typical of the mutant. All these data strongly suggest that LA30A possesses a ts defect in the viral envelope glycoproteins. If the mutant undergoes phenotypic mixing with Rous-associated viruses (RAV), the ts defect is stabilized even when infecting cells which lack receptors for RAV envelope glycoproteins, The possible reasons for this effect are discussed.

Alterations in membrane polypeptides of chick embryo fibroblasts induced by transformation with avian sarcoma viruses

Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive ( ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36°, but at 41°, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3). From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,000 daltons in cell membranes were concluded to be closely correlated with transformation of CEF.

Comparative chemical properties of avian oncornavirus polypeptides

Chemical studies of the four major internal polypeptides p27, p19, p15, and p12 from avian myeloblastosis virus (AMV) and the Prague strain of Rous sarcoma virus (PR-RSV) are presented. Tryptic peptide mapping studies show that the individual polypeptides of AMV possess unique amino acid sequences with no detectable regions of sequence shared by any two polypeptides. Comparisons of the amino acid compositions, amino termini, and tryptic peptide profiles of AMV and PR-RSV polypeptides indicate that analogous polypeptides possess similarities in structure which can be correlated with their antigenic properties.

Nucleic acids of RNA tumor viruses: Identification and ultrastructure

Nucleic acids from both MC29 myelocytomatosis virus and Rous sarcoma virus, Prague strain (RSV-Pr) were spread with the Kleinschmidt technique, examined with the electron microscope, and found to consist of a mixture of both DNA and RNA molecules. The DNA strands possessed length-characteristics of 6–12 μm. RNA molecules, on the other hand, were much shorter and were extended only under the denaturing conditions of both urea and formamide. These molecules were found to be 0.25–2.15 μm in length.

Strain specificity of changes in adenylate cyclase activity in cells transformed by avian sarcoma viruses

Chick embryo fibroblasts transformed by avian sarcoma viruses, B77, the Prague strain of Rous sarcoma virus (PR-RSV), and the Schmidt-Ruppin strain of RSV (SR-RSV) had lower activity of adenylate cyclase, but the same activity of cyclic AMP phosphodiesterase as control cells. The cells infected with their temperature-sensitive mutants in the transforming capacity also had reduced cyclase activity when grown at 36°, but normal activity when grown at 41°, showing a close correlation between viral transformation and cellular adenylate cyclase activity.The characteristics, Km value for ATP, Mg2+ ion dependency, and sensitivity to NaF, of the adenylate cyclase activity reduced by viral transformation varied with the viral strain. It was concluded that the changes of the enzyme activity on transformation were specific to the virus strain used and were not related to the subgroup of virus.

Nuclear localization and covalent linkage of infective virus DNA to chromosomal DNA of non-producer Rous sarcoma cells.

SUMMARY The nuclei of Rous sarcoma cells were prepared from an established line of non-producer rat XC cells transformed with a Prague strain of Rous sarcoma virus (PR-RSV). Electron microscopic examination of the nuclear pellet showed a slight contamination with cytoplasmic debris and an absence of mitochondria. The DNA samples extracted from isolated nuclei and from whole XC cells were both assayed for infectivity in chicken cell cultures and found to contain about the same number of infective units per unit weight of DNA. Furthermore, the DNA from whole XC cells was set free by alkali and sedimented through an alkaline glycerol gradient in order to separate cellular DNA species according to sedimentation velocity. Under these conditions the infective RSV DNA consistently sedimented with the chromosomal 110S DNA and thus behaved as if covalently linked to the chromosomal DNA of XC cells. These results show that the infective virus DNA of non-producer RSV-transformed cells is carried in these cells as an integral part of the cellular chromosome.

Virus productive transformation of marsupial cells by Schmidt-Ruppin strain of RSV.

Rous sarcoma virus (RSV) transforms different species of mammalian cells. The RSV genome is usually not fully expressed in such cells and the virogenic type of virus-cell interaction is obtained1,2. One exception was, however, noted by Svoboda and Klement3 in the case of some hamster tumours induced by Prague strain of RSV (PR RSV) which were found to produce traces (about 1 ID50 per 2.5 g tumour tissue) of infectious RSV.

RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus

Gel electrophoresis of the dissociated 60–70 S RNA prepared from cloned avian sarcoma viruses showed a single major peak corresponding to size class a. Class b RNA was not detectable in these preparations. Class a RNA derived from the Schmidt-Ruppin strain of Rous sarcoma virus had a slightly but reproducibly lower electrophoretic mobility than class a RNA of Prague strain Rous sarcoma virus. The presence of avian tumor virus group specific ( gs) antigens in chicken cells before infection did not detectably influence the electropherograms of the dissociated avian sarcoma virus RNA: cloned sarcoma virus from gs positive and gs negative cells showed identical gel patterns. However, two avian sarcoma virus clones derived from a clonal colony of transformed cells contained a new RNA component which migrated slower than class a RNA. This unusual component was found consistently in the original clonal cultures but was not seen if new cells were infected with the same virus, suggesting a cell mediated modification of viral RNA. Additional evidence was obtained for the absence of class a RNA from avian tumor viruses which cannot form foci in fibroblast cultures. Several leukosis and transformation defective viruses which had not been analyzed previously showed class b RNA only. No significant differences between the class b RNA preparations obtained from several independently isolated transformation defective viruses were detected.

Characteristics of two new avian tumor virus subgroups

Avian tumor viruses capable of growing in cells resistant to the A and B subgroups can be assigned to two new subgroups, C and D. Members of subgroup C are virus B77, Rous-associated viruses types 7 and 49, and a clone-purified line of Prague strain Rous sarcoma virus. These agents plate with equal efficiency in types C O , C A , C B , and C AB chick embryo fibroblast cultures but are excluded from type C BC chicken cells. Rous-associated viruses of subgroup C interfere with sarcoma viruses of that subgroup but do not interfere with sarcoma viruses of subgroups A and B. Conversely, subgroup A and B Rous-associated viruses are unable to interfere alone or in combination with challenge viruses of subgroup C. Neutralization tests and fluorescent antibody staining indicate immunological relatedness of subgroup C viruses and antigenic distinctness from subgroups A and B. Of the subgroup C sarcoma viruses only B77 is oncogenic in hamsters. Members of subgroup D are cloned derivatives of the Schmidt-Ruppin and Carr Zilber strains of Rous sarcoma virus, Carr Zilber-associated virus, and Rous-associated virus type 50. These viruses form foci in C O , C A , C B , C AB , and C BC chick embryo fibroblast cultures. However, their plating efficiency is significantly reduced in all cell types resistant to subgroup B. All subgroup D sarcoma viruses are highly oncogenic for the newborn hamster. The associated viruses are capable of interfering with the sarcoma viruses of subgroup D, but this interference is not seen in all genetic types of chicken cells. The associated viruses of subgroup D also interfere with subgroup B sarcoma viruses, and subgroup B Rous-associated virus induces resistance to subgroup D. Interference does not take place between subgroups A or C and subgroup D. The members of subgroup D are also related immunologically, as indicated by the high incidence and degree of cross reactions in neutralization and fluorescent antibody tests. It appears that the previously described subgroups A and B represent mainly avian tumor viruses of the Western Hemisphere, whereas subgroups C and D are largely composed of viruses propagated in European laboratories.