Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

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.     

Revertants of temperature-sensitive mutants of reovirus: evidence for frequent extragenic suppression.

Twenty-eight independently isolated, spontaneous revertants isolated from temperature-sensitive (ts) mutants of reovirus type 3 representing all the known mutant groups, were examined to determine whether they were intragenic revertants or contained extragenic suppressor mutations. Analysis of the progeny of backcrosses of the revertants to wild type, showed that 25 of the 28 revertants contained is lesions. This result indicated that 25 of the 28 revertants were suppressed pseudorevertants with the suppressor mutation in a gene that could be separated from the parental is lesion by recombination. The nature of the is lesion(s) was examined for a number of the is clones derived from back-crosses. In every case, except one, the parental is lesion was found. In five of the ten suppressed pseudorevertants examined, nonparental is lesions could also be rescued. Two of the nonparental is lesions were in the previously defined recombinant groups. Five of the nonparental is lesions represented a new recombination group or groups since they recombined with the prototype mutants of all of the defined recombination groups. Recombination analysis indicated that the five new mutants fall into two recombination groups for which we propose the designations H and I. The nonparental is lesions rescued from suppressed pseudorevertants may represent suppressor mutations with is phenotype. However, the majority of the suppressor mutations identified had no temperature phenotype and were identified only by their effect on the phenotype of the original is lesion. The fact that a large proportion of revertants were suppressed by extragenic suppressor mutations suggests that mutation events leading to extragenic suppression occur at a much higher frequency than do intragenic events leading to revertant phenotype. These results indicate a general mechanism by which RNA viruses can bypass the effects of deleterious mutations in the absence of intramolecular recombination.     

A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D, and E to genome segments

The double-stranded RNA (dsRNA) of recombinants derived from crosses of the dsRNA-negative, temperature-sensitve (ts) mutants of reovirus type 3 and reovirus serotypes 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments containing the ts lesions. In this way the location of the mutation of the group C prototype mutant tsC(447) os genome segment S2, that of the group D prototype mutant tsD(357) is genome segment L1, and that of the group E prototype mutant tsE(320) is genome segment S3. In addition the location of the temperature-sensitive lesion of serotype 2 is genome segment S1.     

Antibody to virion structural proteins in mammals bearing avian sarcoma virus-induced tumors.

The production of antibody to virion structural proteins was examined in rabbits and hamsters bearing tumors induced by avian sarcoma virus (ASV). Antibody activity was analyzed by the immunoprecipitation of polypeptides from preparations of radiolabeled ASV virions. Antipolymerase and antiglycoprotein activities were monitored by inhibition of the enzyme activity of virion polymerase and by neutralization of focus formation by group D ASV, respectively. All sera from hamsters bearing primary ASV-induced tumors had antibody against the viral gs antigens, but no antipolymerase or antiglycoprotein activity was detectable. Most sera from hamsters bearing tumors induced by injection of cloned hamster tumor cells displayed anti-gs activity, and in addition, some sera exhibited anti-polymerase activity as well. All sera from rabbits bearing primary tumors contained antibody against the gs antigens and the virion polymerase, and some of these sera were shown to have antibody against the virion glycoprotein, gp85, as well.     

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.     

Replication of simian virus 40 in simian virus 40-transformed hamster kidney cells induced by mitomycin C or 60Co gamma irradiation.

Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with ⁶⁰Co γ irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluoresence techniques, respectively. Only some cells in induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or ⁶⁰Co γ irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of γ irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication.     

Biological and biochemical studies on the inactivation of avian oncoviruses by ultraviolet irradiation.

The effects of ultraviolet (uv) irradiation on transforming and replicating capacities of avian oncoviruses and on the synthesis of virus specific products after infection with irradiated virus were studied. Different strains of nondefective avian sarcoma viruses were inactivated at the same rate following single-hit kinetics. The 37% survival dose D₃₇ (1/e) was 736 erg mm^?2 on average. A comparison of the inactivation kinetics in a focus assay (transforming capacity) and an infectious center assay (replicating and transforming capacity) showed no partial inactivation of the virus genome; focus and infectious center formation were inactivated at the same rate. Similar results were obtained when the replicating capacity of the avian sarcoma virus was measured in a plaque assay; focus and plaque formation were inactivated at the same rate. No repair of the uv damage by either complementation or recombination with exogenous or endogenous avian leukosis virus could be demonstrated. The rates of inactivation of avian sarcoma virus assayed in focus and infectious center tests on chick embryo fibroblasts expressing or not expressing chicken helper factor, on chick embryo cells preinfected with RAV-1, and on Peking duck cells were identical. Nondefective avian sarcoma virus and deletion mutants of avian sarcoma virus defective for replication or transformation were inactivated at the same rate. Biochemical analysis of the DNA extracted from a Japanese quail tumor cell line (QT-6) 26 hr after infection with irradiated avian sarcoma virus strain B77 showed a decrease of total virus specific DNA and of full-length covalently closed circular (form I) viral DNA synthesis with increase of the uv dose. Virus-specific RNA synthesis, measured by hybridization of labeled RNA extracted from chicken embryo fibroblasts infected with irradiated virus to viral DNA, and particle production, assayed by uridine incorporation, were also inhibited with increasing uv dose. The inactivation rates for virus-specific DNA and RNA synthesis and for particle production were very similar, but lower than the rate for the loss of infectivity.     

Calicivirus proteins in infected cells: evidence for a capsid polypeptide precursor

Five virus-specific nonstructural proteins were observed in calicivirus-infected cells in addition to the 60,000-dalton capsid polypeptide. They are designated P135, P80, P40, P35, and P29, where the numbers refer to molecular weights in kilodaltons. A new protein, P86, was seen when cells were labeled at 43°. A subsequent 2.5-hr chase at 37° suggested that this protein is a precursor to the capsid polypeptide. Tryptic peptide analysis substantiated this precursor-product relationship. P86 may represent the primary translation product of the 22 S subgenomic mRNA.     

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.     

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.     

RNA specific for the transforming component of avian erythroblastosis virus strain R.

Avian erythroblastosis virus strain R (AEV) contains two species of RNA, 35 S and 30 S, when rescued from nonproducing AEV-transformed chicken cells as pseudotypes of Rous-associated virus-1 (RAV-1) or of AEV-associated virus (REAV). Biological and electrophoretic data suggest that the 30 S RNA is specific for the transforming component of AEV. In fingerprint analysis of RNase T₁-resistant oligonucleotides the 30 S RNA of AEV(RAV-1) yields 13 distinct oligonucleotide spots, 7 of which are unique to the 30 S RNA of AEV, the others are common to RAV-1. The fingerprint of 30 S RNA obtained from AEV(REAV) revealed a similar pattern of unique and shared oligonucleotides. These data suggest that the genome of AEV strain R contains specific genetic sequences which may code for a product that causes transformation of the host cell.     

Epstein-Barr virus-induced proteins. II. Analysis of surface polypeptides from EBV-producing and -superinfected cells by immunoprecipitation

Cells of the Epstein-Barr virus (EBV)-producing line P3HR-1 induced by the tumor promoter TPA and NC37 cells Superinfected with P3HR-1 EBV were surface labeled with ¹²⁵I by the lactoperoxidase method and analyzed by immunoprecipitation with human VCA⁺ MA⁺ sera for virus-induced cell surface polypeptides. Two dominant polypeptides with molecular weights of 80,000 and 250,000 were specifically precipitated. In addition, only traces of polypeptides with 130,000 and 140,000 MW were identified on P3HR-1-EBV-producing cells. The surface of superinfected NC37 cells contained two polypeptides of 80,000 and 140,000 molecular weight. Our experiments demonstrated that the synthesis of the 140,000 polypeptide is Ara C sensitive, while the 80,000 polypeptide is Ara-C insensitive. Both polypeptides were found to be identical in size with [³⁵S]-methionine- and ¹²⁵I-labeled 80,000 and 140,000 envelope polypeptides from purified virus particles. These results may indicate that the identified polypeptides carry antigenic determinants of the EBV-induced membrane antigen (MA) complex.     

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.     

Class II defective avian sarcoma viruses: Comparative analysis of genome structure

The genomes of class II avian sarcoma viruses PRCII, PRCII-p, PRCIV, and Fujinami sarcoma virus (FSV), were studied by oligonucleotide fingerprinting, heteroduplex mapping, and nucleic acid hybridization. All of these viruses are genetically defective and have a small RNA genome between 4.5 and 6.1 kilobases (kb) in length. They contain helper-related sequences at both the 5′- and 3′-ends, but most of the retroviral sequences in the middle of the genome are deleted. In place of this deleted information, a contiguous stretch of transformation-specific sequences, termed fps, is found. These putative oncogenic sequences are about 1.2 kb in PRCII, and those in PRCII-p and PRCIV are roughly 2.9 kb. From the analysis of oligonucleotides, it appears that the fps sequences of PRCII represent a subset of those of PRCII-p. Most of the additional sequences present in PRCII-p but absent from PRCII are at the 5′-half of fps. The helper-related sequences in PRCII and PRCII-p are almost indistinguishable, except that PRCII-p contains slightly more retroviral information at the 3′-end of the genome. Therefore, it is possible that PRCII has been derived by deletion from PRCII-p. By contrast, PRCII-p and PRCIV were found to contain identical fps sequences, but their helper-related sequences have diverged substantially. These two sarcoma viruses either represent two independent isolates or, if derived from a single isolate, they have undergone extensive mutation and recombination with diverse avian retroviruses. FSV was found to differ to a greater extent from other class II sarcoma viruses in both helper-related and fps sequences. The difference in fps sequences is localized in the 5′-half of that region. Considering the variation in fps among all members of class II avian sarcoma viruses, it appears that the 3′-half of that genetic region is more conserved than the 5′-half.     

Replication and plasmid-bacteriophage recombination I. Marker rescue analysis

Recombinant DNA plasmids of pBR322 and T7 DNA were constructed and tested for the presence of various T7 genes. A plasmid containing part of T7 gene 5 (DNA polymerase) was used to transform various strains of Escherichia coli, including a dna B mutant. [³H]Thymidine incorporation and a new method of copy number analysis showed that plasmid DNA was not degraded after infection by T7 as is the E. coli host DNA. Marker rescue between recombinant T7 plasmids and mutant infecting bacteriophage was quantitated by determining the percentage of wild-type progeny phage produced after infection. Replication of the plasmid DNA and infecting phage DNA was controlled independently by a dna B mutation in the host and by gene 5 mutations in the phage, respectively. Marker rescue frequencies decreased slightly, if either plasmid or phage replication was blocked. However, marker rescue dropped below detectable levels, if neither the plasmid nor the phage could replicate. These results show clearly that replication plays a role in plasmid-phage recombination, and possible roles for replication in this process are discussed.     

Biogenesis of vaccinia: evidence for more than 100 polypeptides in the virion.

The polypeptides of vaccinia were separated and analyzed by two-dimensional (2-D) gel electrophoresis patterned after that of O'Farrell (1975). Following labeling with [³⁵S]-methionine, [³³P]phosphate, or [³H]glucosamine, pure virions were dissociated and subjected to electrophoresis using either isoelectric focussing or nonequilibrium pH gradient conditions in the first dimension and SDS-polyacrylamide slab gels in the second dimension. By this means at least 111 spots, of which 7 or more were basic proteins, were resolved in the autoradiograms. Authenticity of several single spots was established. This included a glycoprotein of molecular weight 34,000 (34 K) labeled with [³H]glucosamine; a phosphorylated basic protein of about 11 K marked with ³³PO₄; isolated purified surface tubular elements of 58 K; two major core polypeptides of 60 and 62 K derived from larger precursors after proteolytic cleavage; a precursor polypeptide of 25 K known from previous studies with a ts mutant 1085 to undergo cleavage; and an 18 K polypeptide which appears in wild type and ts 1085 infections under circumstances permissive for cleavage. The 2-D analysis therefore reveals that poxviruses are in terms of their polypeptides, even more complex than had been anticipated previously.     

Cell-free translation of avian erythroblastosis virus RNA yields two specific and distinct proteins with molecular weights of 75,000 and 40,000

Avian erythroblastosis virus (AEV) 28 S virion RNA was translated in vitro in cell-free reticulocyte lysates. Two AEV-specific proteins, one of 75,000 (p75) the other of 40,000 (p40) molecular weight, were detected. p75 is a fusion protein containing gag-specific and AEV-specific peptides. It appears to be translated from the 5′-end of the 28 S AEV RNA and is indistinguishable from the p75 detected in AEV-transformed cells (Hayman et al., 1979). p40 does not share sequences with any viral structural protein. It also contains peptides distinct from those of p75, but one of the five identifiable p40 peptides comigrates with one of the p75 peptides. p40 is translated from a 20 S RNA which contains the 3′-half of the AEV-specific sequences of the genome. These two proteins account for all of the coding capacity of the AEV-specific gene sequences in the 28 S AEV RNA and are candidates for leukemia-specific transforming proteins.     

Studies on the amino acid sequence content of proteins specified by the gag and pol genes of avian sarcoma virus B77.

Data are presented which show that clusters of aggregated adenovirus type 4 give rise to lysis of human red cells. This hemolysis, which is generated by spontaneously formed aggregates of purified virus particles, is inhibited by treatment of the complexes with typespecific antibody. The lysis can also be inhibited by adding negative liposomes to the mixtures. Contrasting with these findings are results which show that hemolytic complexes are produced when particular concentrations of antibody join with suspended adenovirus type 5 particles, harmless by themselves, to form clusters. These aggregates are neutralized by positive liposomes, whereas negative ones are ineffective.