Low freqeuncy production of recombinant subgroup E avian leukosis viruses by uninfected V-15B chicken cells.

15_B chicken contains V-15_B an allele for the spontaneous production of a noninfectious avian leukosis virus, 15_B-ILV. In this paper we present evidence that uninfected 15_B cells also produce subgroup E viruses, designated 15_B-E viruses. In contrast to 15_B-ILV which occurs at an estimated frequency of one event per 10³ 15_B cell days, 15_B-E viruses occur at estimated frequencies of one event per 2 × 10⁹ cell days. The frequency of occurrence of 15_B-E viruses was affected by the level of expression of V-15_B and the presence of endogenous viral alleles which constitutively express subgroup E envelope antigens. The proteins of nine independent isolates of 15_B-E viruses were analyzed by SDS-polyacrylamide gel electrophoresis. All isolates had p27 proteins with the distinctive electrophoretic mobility of the p27 of RAV-0, an endogenous virus. Each isolate had characteristic pairs of p19 related proteins which were similar to pairs of p19 related proteins, which are generated by recombination of PR-RSV-C and RAV-0. Since 15_B-E isolates occurred in uninfected cells and contained proteins which are characteristic of RAV-0 and recombinants of RAV-0, we suggest that 15_B-E viruses are recombinants of endogenous virus information. Since the level of expression of V-15_B and the presence of endogenous viral alleles which constitutively express subgroup E envelope antigens affected the frequency of occurrence of 15_B-E viruses, we further suggest that 15_B-ILV and RNAs which contain information for subgroup E envelope antigens are the parents of 158E viruses.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

Phosphorylation of ribosomal proteins by ribosome-associated protein kinases of Trichosporon cutaneum

Four ribosomal proteins of Mr 13 kDa, 15 kDa, 19 kDa and 38 kDa were identified as phosphorylation substrates for protein kinases tightly associated with Trichosporon cutaneum ribosomes. It was found that proteins of 13 kDa, 19 kDa and 38 kDa were phosphorylated by multifunctional casein kinase II while the protein of 15 kDa by casein kinase I. Proteins of 13 kDa and 38 kDa were detected in the large subunits while 15 kDa and 19 kDa in the small ribosomal subunits. By using isoelectrofocusing the protein of 13 kDa was resolved into two individual phosphorylated forms. The phosphorylation level of both forms was much higher in ribosomes from the cells collected at the exponential growth phase than in those from the stationary phase. The same phosphoprotein forms were identified in ribosomes from in vitro and in vivo [³²P]-labelling experiments.     

Electrophoretic analysis of the RNA of avian tumor viruses

The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37°, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 × 10⁶ daltons and at least one minor RNA species of slightly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, RAV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of Schmidt-Ruppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions.     

Subgroup-specific antigenic determinants of avian RNA tumor virls structural proteins: analysis of virus recombinants.

Two nonglycosylated structural proteins of avian RNA tumor viruses, p15 and p19, were examined for the presence of subgroup-specific antigenic determinants by using competition radioimmunoassays. A comparison of viruses from subgroups A, B, and C revealed that subgroup B and C virus proteins were equally efficient in inhibiting the homologous radioimmunoassays which used antiserum against B77 and iodinated p15 and p19 from B77. On the other hand, subgroup A virus proteins were less efficient than subgroup C viruses in causing inhibition in these assays. That these differences in inhibition were due to true immunological differences was confirmed by using heterologous competition radioimmunoassays. Since it was possible to distinguish the p19 or p15 of subgroup A viruses from the corresponding proteins of either subgroup B or subgroup C virus, recombinant viruses from crosses between leukosis viruses of subgroup A and sarcoma viruses of subgroups B or C were examined. The recombinant viruses have the envelope glycoprotein gene (env) of the subgroup A virus and the sarcoma gene (src) of the subgroup B or C virus. The results show that 14 clones out of 17 examined had p19 from the sarcoma virus. RNA fingerprinting has shown that the src gene is located close to the 3′ end of the genome and is closely followed towards the 5′ end by the env gene. The observed linkage between src and p19 can be explained by postulating that the gene for p19 is located close to the 5′ end of the genome and that recombination takes place between circular forms of the virus genome.     

The induction of antibody reactivity to endogenous viral glycoprotein in 15l5 × 72 [chf(+), V(+)] chickens

Experiments were undertaken to assay for the induction of antibody reactivity to endogenous viral glycoprotein of subgroup E in 15I₅ × 7₂ chickens infected with Pr-A sarcoma virus. Antibody in the sera of the sarcomatous chickens was marginally reactive to the envelope glycoprotein of exogenous viruses of subgroups other than A, but more strongly reactive to the envelope glycoprotein of both the subgroup A infecting virus and the endogenous tumor virus RAV-0. This pattern of reactivity was distinct from the patterns determined in earlier studies with Pr-A-infected chickens which differed from 15I₅ × 7₂ in expression of endogenous viral envelope-related glycoprotein.     

The role of the tvb locus in genetic resistance to RSV(RAV-O).

Genetic susceptibility to RSV(RAV-0), an avian sarcoma virus belonging to subgroup E. has been studied in two sets of inbred chicken lines. Data from the Reaseheath lines suggested that a recessive gene, independent of susceptibility to subgroup B, controlled resistance to subgroup E virus. This gene was assumed to be a new tve locus. Studies of the Regional Poultry Research Laboratory (RPRL) lines suggested that a recessive gene for resistance was located at the tvb locus and thus genetically linked to resistance to subgroup B virus. Crosses and backcrosses of Reaseheath and RPRL lines were assayed for susceptibility to RSV(RAV-0) and RSV(RAV-2) on the chorioallantoic membrane. The data completely support the hypothesis that a single locus controls recessive resistance to subgroup B and E viruses. Therefore, there was no evidence for an independent tve locus.     

The induction of antibody reactivity to endogenous viral glycoprotein in 151(5) X 7(2) [chf(+), V(+)] chickens

Experiments were undertaken to assay for the induction of antibody reactivity to endogenous viral glycoprotein of subgroup E in 15I₅ × 7₂ chickens infected with Pr-A sarcoma virus. Antibody in the sera of the sarcomatous chickens was marginally reactive to the envelope glycoprotein of exogenous viruses of subgroups other than A, but more strongly reactive to the envelope glycoprotein of both the subgroup A infecting virus and the endogenous tumor virus RAV-0. This pattern of reactivity was distinct from the patterns determined in earlier studies with Pr-A-infected chickens which differed from 15I₅ × 7₂ in expression of endogenous viral envelope-related glycoprotein.     

Phosphoproteins, structural components of rhabdoviruses

Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with ³H-amino acids and ³²P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.     

Role of RAV-0 genes in the permissive replication of subgroup E avian leukosis viruses on line 15Bev1 CEF

Rous associated virus type-0 (RAV-0) is a replication-competent endogenous virus of chickens which grows more efficiently on chick embryo fibroblasts (CEFs) from line 15B chickens than on CEFs from line K28. Differences in viral growth on these two sources of cells have been attributed to an early event in the retrovirus life-cycle, at or before viral DNA synthesis. Five in vitro constructed avian leukosis viruses (ALVs) as well as RAV-0 (a subgroup E ALV), RAV-1 (subgroup A), and RAV-2 (subgroup B) have been assessed for their relative growth on 15Bev1 and K28 CEFs. More efficient replication on 15Bev1 CEFs than on K28 CEFs was determined by subgroup E-encoding sequences in env. Subgroup A and B envelope sequences as well as viral LTR, gag, and pol sequences did not obviously bias relative rates of viral replication on the two cell types. We suggest that the unusually permissive replication of subgroup E viruses on 15B CEFs is a receptor-mediated phenomenon and that the line 15B receptor for subgroup E ALVs is more efficient than that of line K28.     

Infection of chick cells by subgroup E viruses.

Studies were carried out to determine whether there is a restriction of replication of the endogenous chicken leukosis virus Rous associated virus type O (RAV-0) in chick embryo fibroblasts (CEF) beyond that imposed by the known cell surface barrier. Following either Sendai virus mediated fusion of RAV-O with surface resistant CEF ($\text{C}\text{E}$ cells), or infection of CEF lacking the surface barrier ($\text{C}\text{O}$ cells), a quantitative 10³- to 10⁴-fold restriction in replication was noted in comparison with RAV-60, a recombinant leukosis virus bearing the same subgroup E envelope as RAV-0 The failure of this internal restriction to operate against subgroup E leukosis viruses was investigated in detail in a series of cloned subgroup E sarcoma virus recombinants isolated following mixed infection with RAV-0 and Prague strain of Rous sarcoma virus subgroup C (PR-C) (i.e., two factor crosses) or following PR-C infection of chf⁺ CEF. RNA from one of these PR-E clones was shown to contain a nearly full complement of RAV-0 specific and RSV specific nucleotide sequences, but that isolate and six others were all free of the restriction observed with RAV-0 replication. One clone may be subject to some restriction. Thus some genetic function(s) of RAV-0 lost or inoperative in recombinant viruses appears important for this restriction. Since RAV-0 can replicate to a “normal” titer in some CEF from particular lines of chickens (line 7, line 100 × 7, and line 15), but apparently not in embryo culture from chicken flocks used in these studies, some cell function(s) also appears important for this restriction.     

A study of the relationship of reticuloendotheliosis virus to the avian leukosis-sarcoma complex of viruses

Reticuloendotheliosis virus (REV) was compared on biological and biochemical grounds with members of the avian leukosis-sarcoma complex of viruses (ALSV). Stocks of REV contained no virus which produced interference with any subgroup of ALSV. REV was unable to complement various strains of Rous sarcoma virus (RSV) as measured by the absence of broadening of host range, “helper” virus activity with the defective Bryan high titer strain of RSV, or the induction of release of RAV-60 from chick embryo cells containing the chick helper factor. Hence, no biological interaction has been detected between REV and the avian leukosis-sarcoma complex of viruses.The RNA structure of the REV was studied using glycerol velocity gradients and polyacrylamide gel electrophoresis. The RNA of REV was distinctly like that of RAV-49, a subgroup C avian leukosis virus. The native RNA of REV recovered after phenol extraction cosedimented with the RNA of RAV-49, and after heat denaturation, both RNAs showed the reduced sedimentation velocity and increased rate of migration in polyacrylamide gel which is characteristic of the tumor virus RNAs.Proteins of REV were examined on SDS discontinuous gel electrophoresis. Five major proteins were detected, two of which were glycoproteins. Coelectrophoresis of REV with avian sarcoma virus B₇₇ showed no polypeptides that were identical in electrophoretic mobilities. In addition, lactoperoxidase catalyzed iodination was employed to selectively label the surface proteins of REV. Two surface proteins were detected; these corresponded to the two viral glycoproteins.     

Radioimmunoassay for the envelope glycoprotein of subgroup E avian leukosis-sarcoma viruses.

The envelope glycoprotein of subgroup E avian leukosis viruses (gpE) was purified from Rous-associated virus type 0 (RAV-0) and used in double-antibody competition radioimmunoassays (RIA) and radioimmune precipitations (RIP). When embryo extracts from various inbred lines of chickens were tested, the results of assays for chick helper factor (chf) and RAV-0 were in complete phenotypic agreement with those of RIA for gpE. The expression of genes at the gs and gp loci varied among inbred lines. Normal line 15I₅ embryos contained gpE but not group-specific (gs) antigens, whereas extracts from virus-free lines 15₁ and 6₁ contained both classes of antigen. Line 15_B embryo extracts were negative for both gpE and gs antigen. Soluble gpE was found in sera of all chf-positive chickens from RAV-0-free lines. Sera from adult line 15_B birds were categorized as: (1) positive for RAV-0 but negative for antibody; (2) negative for RAV-0 but positive for gpE and antibody to gpE; and (3) negative for RAV-0, antibody to gpE, and gpE. Evidence of antibody production was completely correlated in RIP of iodinated gpE and serum neutralizations of subgroup E virus. Results of RIP and gpE RIA indicated that half of the sera from nonviremic line 15_B chickens contained antibodies to gpE as well as gpE.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

Characterization of avian erythroblastosis virus p75

We have studied the 75,000-dalton gag-related polyprotein (p75) found in cells transformed by avian erythroblastosis virus (AEV). AEV p75 was found to be phosphorylated but not glycosylated. Phosphorylation occurs at a serine residue(s) present in the p19 portion of the gag sequences contained at the N-terminal end of p75. No other phosphorylation sites were observed in vivo. AEV p75 was not incorporated into the pseudotyped virion particle. An antiserum specific for the non-gag portion of p75 was prepared by hyperimmunizing chickens that had undergone regression of AEV ts34-induced erythroleukemia with AEV-transformed bone marrow cells. Following absorption with disrupted virions, this serum was capable of immunoprecipitating only AEV p75 and none of the proteins seen following in vitro translation of 20–22 S AEV RNA nor the transforming proteins of a variety of other RNA and DNA tumor viruses. AEV p75 did not contain associated protein kinase activities capable of utilizing either ATP or GTP as phosphate donors when p75 was immunoprecipitated by any of the following antisera: rabbit anti-p19, rabbit anti-disrupted Rous-associated virus-2 virion, Rous sarcoma virus-induced tumor-bearing rabbit serum, or chicken anti-p75 serum. AEV p75 also did not contain associated ATPase activity.     

Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII

In cells infected with the replication-defective avian sarcoma virus PRCII a single virus-specific product is detectable, a polyprotein of 105,000 molecular weight (p105). P105 can be precipitated with antisera togag proteins of avian leukosis and sarcoma viruses. By two-dimensional tryptic peptide analysis of [³⁵S]methionine-labeled proteins we have shown that p105 contains peptides of helper viriongag proteins p19 and p27, but not of p15. In addition a number of peptides are present in p105 that are not found in any of the helper virus gene products including gPr95^env and Pr180^gag-pol. These p105-specific peptides are not detectable in the p60^src protein of Rous sarcoma virus (RSV) nor in thegag-related polyproteins encoded by avian myelocytoma and carcinoma viruses MC29 and MH2 or avian erythroblastosis virus AEV. P105 is not detectably glycosylated, but is heavily phosphorylated. In this respect it resembles p60^src of RSV rather than the polyproteins of avian leukemia viruses. Since p105 is the only viral gene product detectable in nonproducing cells transformed by PRCII, this protein may be important in the initiation and maintenance of oncogenic transformation. The nonstructural sequences in p105 would then represent a new class of transforming gene in avian oncoviruses.     

Phosphoproteins of Spring Viremia of Carp virus

Tryptic peptide analyses of the structural polypeptides of Spring Viremia of Carp virus (SVCV) have established that its two principal species of virion phosphoprotein (NS1, NS2) are related to each other. The NS polypeptides can be distinguished by tryptic peptide analyses from the other major structural polypeptides of SVCV, namely L, G, N, and M. Limited proteolysis of these two viral NS polypeptides, labeled in vitro by [γ-³²P]ATP in reactions catalyzed by the virion protein kinase, or in vivo, have shown that the labeled oligopeptides derived from them can be differentiated from each other, suggesting that they differ with respect to their phosphorylation sites. For both in vitro-and in vivo-labeled NS1 and NS2 species the major phosphorylated amino acid is phosphoserine.     

Tyrosine phosphorylation sites common to transforming proteins encoded by Gardner and Snyder-Theilen FeSV

The Gardner and Snyder-Theilen strains of feline sarcoma virus (FeSV) encode 110,000 molecular weight (M_r) polyproteins as their primary translational products. Cells transformed by a variant of Snyder-Theilen FeSV express an 85,000 M_r polyprotein. Single tyrosine phosphorylation sites identified within each of these viral gene products are shown to represent major in vitro acceptors phosphorylated by the polyprotein associated protein kinases. By two-dimensional tryptic peptide analysis, these acceptor sites are highly related although exhibit minor differences in map position, while by analytical high-performance liquid chromatography, all three elute at approximately the same acetonitrile concentration (21–23%). The tyrosine acceptor sites in these FeSV-encoded polyproteins have been localized by sequential Edman degradation at a position seven amino acid residues distal to their trypsin cleavage site. The major tyrosine acceptors, two serine phosphorylation acceptors common to the p12 structural components of all three polyproteins, a single major phosphothreonine site, and several minor less well-resolved acceptor sites are shown to be phosphorylated in vivo. In addition to the major tyrosine acceptor site initially phosphorylated in vitro, phosphorylation of secondary tyrosine acceptors is shown to occur to proportionately greater extents when in vitro phosphotransferase reactions are carried out for longer times or at higher temperatures.     

Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44

The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis.     

A nonconditional mutant of Rous sarcoma virus containing defective polymerase.

A nonconditional mutant of Rous sarcoma virus (RSV) cd SE52dPR-C (SE52d), which is a recombinant between PR-RSV-C and RAV-0, has been isolated. It is shed from a clone of transformed quail cells, but is unable to exogenously infect or transform any other avian cells. It contains subgroup C envelope glycoproteins and the internal structural protein p27o characteristic of RAV-0. SE52d virions contain no detectable polymerase activity as measured in either an endogenous reaction or after addition of exogenous template. The genome of SE52d contains a deletion of approximately 10% of the genome as compared to PR-RSV, which can be localized between about 0.7 and 0.8 of the genome length from the 3′ terminus of the RNA. The deletion includes about 30% of the polymerase gene. The virions contain no α or β polymerase subunits (95,000 and 65,000 daltons), but do appear to have a small amount of a new protein species of about 57,000 daltons which precipitates with antibody to viral polymerase.