Genome structure of the defective avian sarcoma virus PRCIV

The genome of PRCIV, a defective avian sarcoma virus of class II, was studied by nucleic acid hybridization and oligonucleotide mapping. It has a length of about 6.1 kilobases (kb). A stretch of retrovirus replicative sequences encompassing part of the gag, all of the pol, and most of the env region, in all about 5 kb, is deleted. In its place a 2.9 kb substitution that is transformation-specific for PRCIV was found. Flanking the PRCIV-specific substitution are sequences shared with the helper virus, a stretch of 2.0 kb on the 5′-side, including part of the gag region, and a stretch of up to 1 kb on the 3′-side, including the C-region and possibly some env sequences. A PRCIV-specific cDNA probe representing the transformation-specific sequences was prepared. This probe hybridizes only with RNAs of other class II sarcoma viruses but fails to hybridize with either class I or class III sarcoma viruses.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Chemical crosslinking of proteins in avian sarcoma and leukemia viruses

We have investigated the nearest neighbor relationships of proteins in avian sarcoma and leukemia viruses by means of bifunctional crosslinking agents. In intact virions dimethyl suberimidate and dithiobispropionimidate induce the formation of covalently linked multimeric species of all four internal structural proteins (gag proteins). By immunological tests and by two-dimensional polyacrylamide gel electrophoresis the major species were identified as homotypic dimers of p27, p19, p15, and p12, and homotypic tetramers of p27. No recognizable heterotypic multimers of gag proteins are formed. We conclude that the major gag protein-protein interactions in virions occur between like species. The crosslinking agents also introduce links into the env protein dimer, gp85-gp37, and form a higher multimer of this dimer.     

Structural similarities of proteins encoded by three classes of avian sarcoma viruses

The structure and location of the phosphorylation sites of a number of avian sarcoma virus polyproteins have been examined by protease cleavage analysis. The PRCIIp and PRCII polyproteins, P170^gag-fps and P105^gag-fps yield indistinguishable cleavage fragments from an N-terminal region of 65,000 molecular weight, including the gag/non-gagjunction. This provides strong support for the view that PRCII arose directly from PRCIIp by a genomic deletion. For P909^agag-yes, P800^gag-yes, and P105^gag-fps the major tyrosine phosphorylation sites are on C-terminal fragments of 27,000, 26,500, and 36,000 molecular weight, respectively. Further similarities have been shown by partial sequence analysis of the tyrosine phosphorylation sites; the positions of trypsin and staphylococcal V8 protease cleavage sites largely correspond in the src, fps, and yes gene products. The homology between the src and yes products is particularly striking. They yield C-terminal V8-resistant fragments of similar size, containing the major tyrosine phosphorylation sites which are indistinguishable after further cleavage with several proteases. These results suggest structural and functional relatedness between the src, fps, and yes gene products despite the lack of hybridization between their DNA sequences.     

Transforming proteins of fujinami and PRCII avian sarcoma viruses have different subcellular locations

The subcellular locations of transforming proteins encoded by the related avian sarcoma viruses, PRCII and Fujinami sarcoma virus (FSV), were compared by cell fractionation and by indirect immunofluorescence. Whereas both viruses encode gag-fps proteins associated with tyrosine-specific kinase activity, FSV is more highly tumorigenic than PRCII in vivo. Cell fractionation studies showed that the PRCII transforming protein, P105, became associated with the high-speed particulate fraction shortly after synthesis. However, PRCII P105 did not fractionate with the plasma membrane marker, but rather with high-density membranes. It is unique in this subcellular localization among viral tyrosine kinases. This membrane association was found to be relatively insensitive to salt concentration and did not require divalent cations. Immunofluorescent studies, using anti-fps serum, showed that the PRCII protein was present in discrete, large, cytoplasmic patches, as well as in a juxtanuclear location. In contrast, FSV-encoded P130 was found to fractionate with the plasma membrane marker when cells were analyzed in low salt in the presence of magnesium. However, at higher salt concentrations and in the absence of magnesium, the bulk of P130 was found to be soluble. Immunofluorescent staining of FSV P130 revealed a diffuse, cytoplasmic pattern that was distinct from that of the PRCII product. The observed difference in the subcellular localization of these transforming proteins may be the cause of the difference in tumorigenicity between the two viruses.     

Temperature-sensitive kinase activity associated with various mutants of avian sarcoma viruses which are temperature sensitive for transformation

Antisera against the transforming gene product of avian sarcoma virus (ASV), called pp60^src, were raised in tumor-bearing rabbits by inoculation of a high dose of the avian sarcoma virus Schmidt-Ruppin strain of subgroup D (SR-D) into newborn animals. Four mutants of ASV with temperature-sensitive defects for transformation have been investigated for temperature sensitivity of the sarcoma gene-associated protein kinase activity in comparison to that of their wild-type parents. The inactivation rate of the protein kinase from the mutants MI 100, OS122, OS538, and NY68 was two-to threefold faster than that of the respective parents which belong to various subgroups of the Schmidt-Ruppin strain, SR-B, SRD, and SR-A. During heat inactivation, dephosphorylation of ³²P-labeled pp60^src immune precipitated from mutant virus-infected cells was not parallel to the inactivation of the kinase. The amount of [³⁵S]methionine-labeled pp60^src precipitated from mutant- and wild type-infected cells was not sensitive to heat treatment. The kinase activity associated with pp60^src of mutant- and wild type-infected cells was protected from inactivation by the presence of sera from tumor-bearing rabbits (TBR) during the heating procedure. Protein kinase activity of MI100 increased up to fourfold during this process, while with other mutant and wild-type kinase activities such a curing by TBR-sera was also observed but not to the same extent. Immune complexes obtained from transformed cells treated with TBR-serum and Staphylococcus aureus were used as a source of kinase(s) to phosphorylate exogenously added cell lysates as targets. Alternatively, [γ-³²P]ATP was added to cell lysates to allow phosphorylation of polypeptides present in the lysates. This procedure resulted in phosphorylation of proteins with molecular weights of 60K, 35K, and 23K which were absent from the normal or leukosis-virus-infected controls. Several proteins of high molecular weight, one of them of 150K, were enhanced. Treatment of these phosphoproteins with TBR-serum resulted in precipitation of the 60K and 23K proteins but not of the 150K and 35K phosphoproteins.     

Transformation parameters of chick embryo fibroblasts transformed by Fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses

The transformation parameters of chicken embryo fibroblasts transformed by replication defective avian sarcoma viruses were compared with those of cells transformed by Rous sarcoma virus (RSV). Two classes of avian sarcoma virus with transforming genes distinct from the src gene of RSV were examined: Fujinami sarcoma virus (FSV), PRCII, and PRCII-p were used as representatives of one class, and Y73 as a representative of the other class. The transformation parameters examined included morphological markers: cell surface morphology, the disappearance of microfilament bundles, and the loss of cell surface fibronectin; alterations in growth properties: growth to high densities and growth in suspension; and metabolic changes: increased uptake of hexose, release of plasminogen activator, and increased synthesis of hyaluronic acid. In general, all of the viruses induced a phenotype which was qualitatively similar to that of RSV-transformed cells, although the extent of each change was less than that observed with RSV. One strain of FSV was found to be coordinately temperature sensitive for the induction of all phenotypic markers examined, whereas another strain of FSV and the other viruses examined were temperature resistant. PRCII-transformed cells had a lower efficiency of colony formation in agar suspension and showed only partial disruption of microfilament bundles; this partial transformation phenotype may be correlated with the low oncogenicity of this virus. These findings in conjunction with previous studies on protein phosphorylation in ASV-transformed cells are consistent with the idea that the replication-defective avian sarcoma viruses transform cells by a mechanism similar to that involved in transformation by RSV.     

Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response

The cellular immune competence of lymphocytes obtained from birds infected with acute or chronic leukemia viruses was studied. The in vitro blastogenic response of thymus-derived lymphocytes (T cells) to the mitogens phytohemagglutinin (PHA) and connanavalin A (Con A) correlates with the ability of birds to elicit a cellular immune response (C. K. Naspitz and M. Richter, 1968, Prog. Allergy12, 1–85; P. Toivanen and A. Toivanen, 1973, J. Immunol.111, 1602–1603). The PHA response of splenic lymphocytes from birds infected with the avian acute leukemia viruses, myelocytomatosis virus strain 29 (MC29), avian erythroblastosis virus (AEV), avian myeloblastosis virus (AMV), or reticuloendotheliosis virus (REV-T), was completely suppressed within 1 week after virus infection of chickens by avian acute leukemia virusus. Spleen cells from chickens infected with the chronic leukemia viruses, Rous-associated viruses (RAVs) types 1 and 3, exhibited PHA responses similar to those of uninfected birds. In contrast, lymphocytes from RAV-2 infected birds had suppressed PHA responses. Acute leukemia viruses are replication-defective and are associated with replication-competent nontransforming helper viruses. When chickens were infected with helper viruses isolated from the acute leukemia virus stocks by endpoint dilution, the T-cell response to various T-cell mitogens was completely suppressed. The helper viruses, therefore, contribute to the immunosuppression which occurs during the development of avian acute leukemia. The rapid lethality of the acute leukemia viruses could be, in part, the result of the immunosuppressive activity of the associated helper viruses. Though all the acute leukemia viruses severely immunosuppress their hosts, the mechanism by which the immunosuppression was induced by these viruses differed. REV-T was the only acute leukemia virus which induced a population of splenic suppressor cells. All the viruses of the leukosis-sarcoma complex tested which had subgroup B specificity were immunosuppressive while those of subgroup A were not. These results suggest that immunosuppression by these viruses may be related to subgroup specificity.     

The transformation-specific proteins of avian (Fujinami and PRC-II) and feline (Synder--Theilen and Gardner--Arnstein) sarcoma viruses are immunologically related

The detection of protein kinase activity associated with transformation-specific gene products of Rous sarcoma virus (RSV), Abelson murine leukemia virus (Ab-MuLV), the Snyder-Theilen (ST), and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) and the recently characterized Fujinami (FSV) and PRCII avian sarcoma viruses suggests that these viruses may induce oncogenic transformation by similar mechanisms. In spite of their similar enzymatic properties, the transformation-specific proteins of RSV or Ab-MuLV were not found to share common antigenic determinants with those of the ST or the GA strains of FeSV. These results suggest that vertebrates contain multiple unrelated genes that code for proteins with associated tyrosine-specific protein kinase activity and oncogenic properties when incorporated into the genome of a retrovirus. In contrast, the transformation-specific proteins of ST and GA-FeSV were shown to be immunologically related to those of the FSV and PRCII avian sarcoma viruses. The antigenic determinants shared by these viral proteins have been mapped within their respective sarcoma virus-specific regions, suggesting that the cellular insertions present in these avian and feline sarcoma viruses are related. These observations indicate that potentially oncogenic sequences have been conserved during the evolution of feline and avian genomes and have been independently acquired by two sets of sarcoma viruses.     

Integration of avian sarcoma virus DNA in chicken cells

Host-range and host-range deletion mutants of type 5 adenovirus belonging to two complementation groups, whose mutational lesions map physically within the left 11% of the type 5 genome, were efficiently complemented in mixed infection by type 12 adenovirus. The complementation of these early function mutants by type 12 is in contrast to the lack of complementation found previously for two other groups of early function mutants, and it is consistent with functional relatedness between products encoded by early region 1 of the respective serotypes. No evidence for recombination between the type 5 mutants and type 12 was found.     

Induction of anemia by avian leukosis viruses of five subgroups

Chickens infected with avian leukosis viruses frequently manifest anemia during the development of disease. Previous work has established that infection of the 8-day-old hatched chick with virus results in the synchronous appearance of a profound anemia. In this communication, we have used this phenomen to test viruses representing five subgroups of avian leukosis for the ability to induce anemia. The results indicate that viruses of B and D subgroups induced severe anemia while viruses of subgroups A, C, and F induced only mild anemia or none at all.     

Isolation of an avian sarcoma virus-specific protein kinase from virus particles

Avian sarcoma viruses (ASV) of the Schmidt-Ruppin strain (SR) contain a protein kinase activity which specifically phosphorylates the IgG of sera from tumor-bearing rabbits (TBR). The amount is comparable with that from transformed cells. The activity is thermolabile in two mutants with a temperature-sensitive lesion in the sarcoma (src) gene. Ion-exchange column chromatography on DEAE-cellulose and phosphocellulose allowed a 250-fold purification of enzymatically active protein kinase with a molecular weight of 60,000 (60K). It phosphorylated casein and the heavy chain of IgG of TBR sera but not of control sera. Phosphorylation of casein could be completely inhibited by TBR serum and resulted in phosphorylation of IgG. Purification of the protein kinase from a mutant virus, OS122, and its wild-type SR-D revealed a threefold higher thermolability for the mutant enzyme. Partial proteolytic digest of the [³⁵S]methionine-labeled 60K protein obtained from the phosphocellulose column by immune precipitation was indistinguishable from that of pp60^STC precipitated from SR-D-transformed cells.     

Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60^src, the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60^src. Furthermore, pp60^src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.     

Characterization of DNA complementary to nucleotide sequences adjacent to poly(A) at the 3'-terminus of the avian sarcoma virus genome.

The temperature-sensitive influenza virus A/Hong Kong/68 (H3N2) ts-1[E] has been used as a prototype live attenuated influenza virus vaccine. Using recently developed techniques to map the genome of influenza viruses and to “genotype” influenza virus recombinants, the temperature-sensitive lesions in the virus were identified. These defects, responsible for the attenuation of the virus, are located in the genes for the P3 protein and the nucleoprotein and are associated with virus-specific RNA synthesis. Hong Kong/68 (H3N2) ts-1[E] virus can also serve as a donor of “attenuation characteristics” for the selection of recombinant strains which have different surface antigens and may be used as vaccine strains in the future. The temperature-sensitive mutations of Hong Kong/68 (H3N2) ts-1[E] virus were previously transferred to recombinant viruses carrying the HO hemagglutinin. The RNAs of 8 of these temperature-sensitive recombinants were analyzed. One of these viruses, R1, classified in group 1 of the Hong Kong mutant virus set was found to possess a ts defect only in the P3 protein. R8, a member of group 2 of the Hong Kong mutant virus set had a ts mutation in the nucleoprotein.     

Antigenic and structural studies on the transforming proteins of Rous sarcoma virus and Yamaguchi 73 avian sarcoma virus

A widely cross reactive antiserum raised against denatured pp60v-src, the transforming protein encoded by Rous sarcoma virus, was used to test antigenic relationships with the transforming gene products encoded by other avian sarcoma viruses. The results showed that P90gag-yes, the transforming protein of a representative of Class III avian sarcoma viruses, is antigenically related to pp60v-src. Tryptic phosphopeptide analysis of P90gag-yes revealed two phosphotyrosine-containing peptides and one phosphoserine-containing peptide. One of the phosphotyrosine-containing peptides comigrated with the phosphotyrosine-containing tryptic peptide from pp60v-src.     

Characteristics of avian sarcoma virus strain PRCIV and comparison with strain PRCII-p

Avian sarcoma virus strain Poultry Research Centre IV (PRCIV) (J. G. Carr and J. G. Campbell (1958)Brit. J. Cancer12, 631–635) is highly oncogenic in chickens and transforms chicken embryo fibroblasts in culture. It is defective in replication, unable to code for complete functional products of the three essential viral genes gag, pol, or env. Nonproducing cells transformed by PRCIV lack the transforming protein of Rous sarcoma virus, pp6^src but synthesize a new transformation-specific protein of 170,000 MW. This P170 contains partial gag sequences, probably at its N-terminus and transformation-specific, cell-derived sequences, probably in the C-terminal portion of the molecule. In two-dimensional tryptic peptide maps P170 of PRCIV is indistinguishable from the transformation-specific protein P170 of the previously described PRCII-p (M. L. Breitman, J. C. Neil, C. Moscovici, and P. K. Vogt (1981)Virology108, 1–12). Both P170 proteins also contain all but one tryptic peptide of the transformation-specific gag-linked protein P105 found in cells transformed by avian sarcoma virus PRCII. The P170 proteins of PRCIV and PRCII-p have an associated tyrosine-specific kinase activity similar to P105 of PRCII. All three agents, PRCII, PRCII-p, and PRCIV, belong to class II of avian sarcoma viruses. In cells transformed by PRCIV or PRCII-p secondary gag-linked transformation-specific proteins of less than 170,000 MW are sometimes seen, but limited in vivo and in vitro passage of the viruses shows the P170 to be a stable characteristic of PRCIV and PRCII-p transformation. The genomic RNA of PRCIV and PRCII-p sediments at 30 S corresponding to a size of about 6.1 kilobases.     

The avian sarcoma virus PRCII lacks 1020 nucleotides of the fps transforming gene

Fujinami sarcoma virus (FSV) and PRCII avian sarcoma virus both encode gag-fps transforming proteins associated with tyrosine-specific protein kinase activity; however, PRCII has a lower oncogenic potential than does FSV. In this study, the genomes of PRCII and FSV have been compared. By hybridization of PRCII [32P]RNA to FSV DNA on Southern blots, a large internal deletion in the 5' half of the fps gene in PRCII has been mapped. To determine the exact size and location of the deletion in PRCII, dideoxy sequencing of PRCII RNA with FSV DNA fragments as primers was used. The FSV sequence corresponding to the deletion in PRCII was flanked by 6-base direct repeats ( AGCTGG ) at 1614-1619 and 2634-2639 nucleotides. One copy of the direct repeat was retained in the PRCII genome. The length of the deleted region was 1020 nucleotides. The deletion in fps did not alter the kinase domain or ATP-binding site of the P105 transforming protein of PRCII. It was shown that the specific kinase activity of P105 was as high as that of FSV P130 . The sequence deleted from PRCII was found to encode part of a large hydrophilic domain. In the accompanying paper [J. Woolford and K. Beemon (1984) Virology 135, 168-180], evidence that the PRCII and FSV proteins have different subcellular locations and solubility properties, possibly due to the loss of this domain, is presented. These alterations in the structure and location of the PRCII protein may prevent it from phosphorylating certain substrates involved in oncogenic transformation.     

Comparative analysis of the enveloped virions of two granulosis viruses of the armyworm, Pseudaletia unipuncta

The enveloped virions of two strains of a granulosis virus, a synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain which infect the armyworm, Pseudaletia unipuncta, were liberated from their capsules with 0.02 N NaOH and purified by filtration through membrane filters. The envelopes were solubilized with 0.1% Triton X-100. The enveloped virions, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), had 9 to 12 polypeptides, 3 of which were associated with the naked virion. The two strains differed in their electrophoretic patterns mainly in the presence of two heavy polypeptides (89,000 and 97,000 daltons) and a 52,000-dalton polypeptide in GVH and in the absence of the heavy polypeptides and the presence of a 57,000-dalton polypeptide in GVO. The two heavy polypeptides were sensitive to proteinase. Two two-dimensional electrophoreses (first dimension with agarose gel electrophoresis and second dimension with SDS-PAGE and immunoelectrophoresis) of the envelope of GVH resolved two polypeptides, 37,000 and 48,000 daltons, which were serologically related to the synergistic factor present in the capsule of GVH; however their molecular weights differed from that of the GVH synergistic factor. In GVO, no polypeptide serologically related to the synergistic factor was detected in the envelope. The enveloped virions purified by differential filtration were infectious when fed to armyworm larvae, but the naked virions, free of envelopes, were not infectious.