Mutants Of Avian Sarcoma Virus Research Articles

Glycosylation effects on cancer development

Changes in glycosylation associated with oncogenic transformation were first demonstrated with glycosphingolipids over four decades ago. Certain gangliosides, GM3, GD1a and GD1b, which are highly expressed on non-transformed cells, were found to be greatly reduced upon oncogenic transformation [1, 2]. A subsequent study using a temperaturesensitive mutant of avian sarcoma virus showed close correlation between reduction of GM3 expression and appearance of transformed cell phenotypes. Further study by transfecting GM3-synthase gene into v-Jun transformed mouse and chicken fibroblast cells introduced an important concept that the oncogenic phenotype of cancer cells can be reverted to normal-like phenotype by manipulating the expression level of certain glycosphingolipids [3]. Many studies along similar lines have since been developed with various types of cells. Numerous studies have demonstrated the close association of various types of N-glycans and O-glycans with cancer development. These findings could potentially be applied to interfere with cancer progression or reversion to normal cell phenotypes. Elevated expression of Nacetylglucosaminyltransferases, such as GnT-III and GnT-V, which are involved in branching modifications of N-glycans, has been observed upon cellular transformation [4]. In addition, the truncated O-glycans known as Tn and its sialylated version Sialyl Tn antigen, are often found to be expressed by many different cancers in immunohistochemical studies, and can arise from altered expression of the enzymes involved in O-glycosylation of proteins [5]. Finally, elevated expression of Lewis-related glycans, such as Sialyl Lewis A on mucin-type glycoproteins of tumor cells has been observed, and is the basis of serum biomarker test for cancer, such as the CA-19-9 antigen [6, 7]. In this specialized volume, entitled “Glycosylation Effects on Cancer Development”, we present some of the recent progress and the development of new concepts in this important and growing area. T. Miyagi and her colleagues describe altered expression of four types of sialidases in cancer cells and focus on Neu3, which has enhanced expression in various cancer cells. Neu3 is known to specifically hydrolyze gangliosides including GM3, but not sialyl groups of other glycans, and is indicated to stimulate cell growth through interacting with growth factor receptors. In addition, the authors discuss their result that the knockdown of Neu3 expression by siRNA causes apoptosis in cancer cells, and suggest Neu3 could be a useful target for inhibition of cancer progression. K. Furukawa’s group summarizes their studies on GD3 and GD2, which are highly expressed in melanomas and small cell lung cancer cells. They found that the gangliosides enhance cell motility and invasiveness by stimulating focal adhesion kinase and Lyn kinase, which phosphorylate paxillin and p130Cas. Further studies using the reconstructed membrane system indicated GD3 and GD2 function in glycosphingolipidenriched microdomains/rafts. This line of study may eventually lead to a novel approach to block cancer metastasis. S. Sonnino and his colleagues report, for the first time, the effect of ionizing radiations on plasma membraneS.-i. Hakomori (*) Division of Biomembrane Research, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA e-mail: hakomori@u.washington.edu

Role of the Constitutive Splicing Factors U2AF65 and SAP49 in Suboptimal RNA Splicing of Novel Retroviral Mutants

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

Genetic selection for balanced retroviral splicing: novel regulation involving the second step can be mediated by transitions in the polypyrimidine tract.

Incomplete splicing is essential for retroviral replication; and in simple retroviruses, splicing regulation appears to occur entirely in cis. Our previous studies, using avian sarcoma virus, indicated that weak splicing signals allow transcripts to escape the splicing pathway. We also isolated a series of avian sarcoma virus mutants in which env mRNA splicing was regulated by mechanisms distinct from those of the wild-type virus. In vitro splicing experiments with one such mutant (insertion suppressor 1 [IS1]) revealed that exon 1 and lariat-exon 2 intermediates were produced (step 1) but the exons were not efficiently ligated (step 2). In this work, we have studied the mechanism of this second-step block as well as its biological relevance. Our results show that the second-step block can be overcome by extending the polypyrimidine tract, and this causes an oversplicing defect in vivo. The requirement for regulated splicing was exploited to isolate new suppressor mutations that restored viral growth by down-regulating splicing. One suppressor consisted of a single U-to-C transition in the polypyrimidine tract; a second included this same change as well as an additional U-to-C transition within a uridine stretch in the polypyrimidine tract. These suppressor mutations affected primarily the second step of splicing in vitro. These results support a specific role for the polypyrimidine tract in the second step of splicing and confirm that, in a biological system, uridines and cytosines are not functionally equivalent within the polypyrimidine tract. Unlike the wild-type virus, the second-step mutants displayed significant levels of lariat-exon 2 in vivo, suggesting a role for splicing intermediates in regulation. Our results indicate that splicing regulation can involve wither the first or second step.

Mutations in 3′-untranslated region of avian sarcoma virus mutant LA46 genome confer the cis-acting temperature-sensitive replication defect

Avian sarcoma virus (ASV) PRCII mutant LA46 carried a unique cis-acting temperature sensitive (ts) defect affecting virus replication. The 3′-untranslated region of the LA46 genome contained multiple base substitutions in the direct repeat region and a long insertion. Although these sequences were not present in PRCII, most of these changes were found in various other ASVs and the long insertion shared extensive homology with the so called E region in certain ASV strains. By constructing chimeric retroviruses, the is defect was mapped to the altered 3′-noncoding region. However, site-specific mutagenization targeted towards a four-nucleotide substitution unique to LA46 failed to implicate these mutations in the is phenotype

Cloned DNA of defective avian sarcoma virus mutant LA46 encodes the cis-acting temperature-sensitive defect in replication

Avian defective sarcoma virus mutant LA46 carries a temperature-sensitive defect in replication and transformation. To elucidate this defect, we cloned the integrated provirus of LA46. By DNA-mediated transfection, the cloned DNA induced fusiform-transformed foci in chick embryo fibroblasts without helper virus. LA46-encoded transformation-specific protein p105 was expressed in these transformants in the absence of helper virus-encoded proteins. Superinfection of the transformed cells with different helper viruses resulted in the resue of pseudotypes. All the rescued pseudotypes retained helper viruses resulted in the resue of pseudotypes. All the rescued pseudotypes suggesting that the defect was due to a cis-acting lesion in the LA46 genome. Restriction enzyme comparison between LA46 and wild-type virus revealed sequence differences near the 5′ and 3′ termini of the LA46 genome, including the long terminal repeat regions.

Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).

Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).

The mitogenic activity of pp60 v-src, the oncogenic protein product of the src gene of avian sarcoma virus, is independent of external serum growth factors

The oncogenic pp60 v-src product of ASV (avian sarcoma virus) is shown to be a potent endogenous mitogen, which, unlike mitogens such as PDGF (platelet derived growth factor), is able to stimulate host cell proliferation without the help of other growth factors. Thus, NRK rat cells, infected with a temperature-sensitive ASV mutant which produces an abnormally thermolabile pp60 v-src became proliferatively quiescent at a pp60 v-src -inactivating 40°C in medium containing either 0.2% calf serum or no serum at all. Adding PDGF stimulated the quiescent tsASV-NRK cells at 40°C to initiate DNA replication in medium containing 0.2% serum, but not in serum-free medium. By contrast, activating internal pp60 v-src by dropping the temperature to a permissive 36°C stimulated these quiescent cells to transit G 1, initiate DNA replication and to enter mitosis even in serum-free medium. Thus, relative to PDGF, endogenous pp60 v-src behaves as a complete mitogen.

Partial characterization of the mitogenic action of pp60v-src, the oncogenic protein product of the src gene of avian sarcoma virus.

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36 degrees C, but at 40 degrees C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v-src product of the virus' transforming src gene. At 40 degrees C, these tsLA23-NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet-derived growth factor (PDGF) at 40 degrees C, or (2) transformed cells by lowering the temperature to a pp60v-src-activating 36 degrees C without adding exogenous growth factor(s). The level of pp60v-src kinase activity rose dramatically in these serum-deprived cells within 30 minutes of lowering the temperature to the permissive 36 degrees C, and it fell just as rapidly when the cells were returned to the restrictive 40 degrees C. As little as a 2-hour exposure to 36 degrees C, with an attendant 2-hour burst of pp60v-src kinase activity, was enough to stimulate serum-deprived tsLA23-NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v-src activity was needed for these serum-deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23-NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v-src-stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v-src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v-src were produced with comparable efficiency, although it took longer for pp60v-src-stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein-synthetic machinery.

Phenotypes of cell hybrids formed by fusion of normal and Rous sarcoma virus transformed cells

Untransformed mouse cells were fused with rat cells transformed by a temperature sensitive mutant of avian sarcoma virus, and cell hybrids were isolated in the absence and in the presence of selective medium. None of the hybrids isolated were as transformed as the parent rat cells. All hybrids isolated in the absence of selective medium showed a phenotype similar to that of the untransformed mouse cell parent. Cell hybrids isolated on selective media, however, were more heterogenous. Some showed a phenotype that were intermediate between that of the two parental cells, while others were more like the untransformed mouse cells.

Analysis of the pathogenicity of transformation defective partial deletion mutants of avian sarcoma virus: Characterization of recovered viruses which encode novel src specific proteins

Several transformation defective ( td) mutants of the Prague strain of Rous sarcoma virus, which had been previously shown to have deletions of varying sizes and positions within the src gene, were tested for their ability to induce disease in chickens. Several of the mutants induced sarcomas after long latency, in particular two mutants which had deletions spanning the presumed active site (i.e., the phosphotyrosine residue) of the RSV transforming protein, pp60 src . Viruses recovered from these tumors, as well as the tumors themselves, were analyzed to study the mechanism of tumor induction. In some examples proviral DNA structurally similar to wild-type virus was found in tumors and virus recovered from these tumors was shown to transform chick cells in vitro. Transformation specific proteins of 55,000 Da immunoprecipitable with antisera against pp60 src were encoded by the recovered viruses. These proteins displayed a protein kinase activity, appeared to have small deletions in the amino termini, and by phosphotryptic peptide mapping appeared to contain novel phosphotyrosine tryptic peptides, when compared to wild-type virus, which were presumably derived from endogenous c-src.

The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells.

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40 degrees C and were rendered quiescent by serum deprivation. These serum-deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40 degrees C or about 9 hours after shifting the cultures to 36 degrees C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum- and temperature-stimulated tsLA23-NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23-NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non-neoplastic cells. At the nonpermissive 40 degrees C, the calmodulin content of the tsLA23-NRK cells dropped to the non-neoplastic level. When these phenotypically nontransformed cells were enabled to reenter the cell cycle while still in low-serum medium by a 40 to 36 degrees C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.

Isolation and characterization of host range mutants of avian sarcoma virus.

Two host range mutants of avian sarcoma virus (ASV) were isolated from a clone of the Bratislava 77 strain, subgroup C (B77-C). An HR- mutant was obtained by adaptation to duck cells and showed almost the same efficiencies of transformation (e.o.t.) and growth in cultures of chick embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF). An HR+ mutant showed lower e.o.t. and slower growth on DEF than on CEF. Characterization of these mutants and wild-type B77-C showed that (i) unlike the host range mutants reported previously, these mutants and wild-type B77-C have common antigenicity, (ii) the number of infective centres is almost the same for the two mutants and the wild-type, and so the adsorption and integration of these viruses are similar, and (iii) the content of viral RNA in DEF infected with HR- is increased and decreased in the case of HR+ compared with the wild-type.

Alterations in the genomes of avian sarcoma viruses

We describe 12 nonconditional mutants of avian sarcoma virus. Eleven were replication defective and one appeared to retain wild-type functions. Comparison of restriction endonuclease digests of viral DNA revealed that 8 of 11 replication defectives contained large deletions of viral information. A functionally wild-type variant contained an insertion of sequences between the env and src genes. The deletions appeared to be randomly distributed throughout the replicative genes: gag, pol, and env. In most, but not all, cases, the phenotype of the deletion mutants could be simply correlated with the location and extent of the deletions. In a few cases, the defects in expression appeared to be more extensive than would be expected from a single point or deletion mutation, suggesting that these mutants contained multiple mutations or a single mutation in a region affecting several viral functions.

Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus.

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.

Fibrin Clot Retraction in Normal and Transformed Avian Fibroblasts<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Normal avian embryo fibroblasts induced retraction of a fibrin clot. The expression of high levels of this activity was associated with the exponential growth phase of the cell division cycle. Cells transformed by avian sarcoma virus (ASV) and other agents lost this activity regardless of the cell cycle phase. Cultures infected with transformation-defective ASV mutants retained a high level of fibrin clot retraction. However, this activity could not be recovered when temperature-sensitive, virus-transformed cells were shifted to a nonpermissive temperature (41 degrees C). The results indicated that, in this cell system, loss of fibrin clot retraction could be considered a reliable parameter of cell transformation.

An avian sarcoma virus mutant which produces an aberrant transformation affecting cell morphology

ST 529 is a temperature-sensitive mutant of Rous sarcoma virus (RSV) strain SR-A, which causes an unusual pattern of phenotypic changes in cells that it transforms. At 35°, ST 529-transformed cells exhibit an elongated, fusiform morphology (morph f), and also possess an aberrant and “unlinked” phenotypic pattern of transformation-related properties. ST529-transformed cells, at 35°, resemble classically transformed cells in respect to density-independent growth, sugar uptake, protease levels, and ability to form soft agar colonies. However, they differ from classically transformed cells in respect to cell morphology, fibronectin levels, adhesiveness, and organization of actin stress fibers. At a nonpermissive temperature (42°), ST529-infected cells appear phenotypically normal. pp60 src isolated from ST529-infected cells at 42° possesses little or no associated protein kinase activity. Kinase activity becomes detectable rapidly, however, within 30 min after a shift to 35°, and reaches maximal levels within 3 hr after the shift. It is probable that an unusual, mutant src gene product is responsible for the novel pattern of transformation-related changes observed in ST529-infected cells. Previous studies utilizing mutants or variants of RSV have suggested that there are probably at least two biologically significant targets for pp60 src . The present experiments provide additional evidence for a multifunctional src gene product.

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the src gene of avian sarcoma virus

We have characterized src proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with 32PO 4 or [ 35S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmus et al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group ( Class I) encoded src proteins of normal size (60,000 M r ); these proteins were hypophosphorylated and exhibited little or no protein kinase activity. Class II mutants displayed immunoprecipitable src proteins of less than normal size. In three cases, the short src related proteins were mapped to the amino terminus of wild-type pp60 src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car☐yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000 M r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car☐yl terminal three-fourths of pp60 src. The smallest group of mutants ( Class III) did not produce detectable src proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60 src. The active proteins larger than pp60 src ranged up to 68,000 M r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functional src protein of 68,000 M r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus of src. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes of src.

Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles.

Mutants of avian sarcoma virus which lack a functional DNA polymerase were found to be nonselective in the incorporation of host cell tRNA's into virus particles. In contrast, mutants which possess a functional DNA polymerase but lack the viral genome RNA contained a specific subset of the host cell tRNA population, indistinguishable from that of the wild-type virus. Thus the reverse transcriptase, and not the viral RNA, is probably the major factor determining which tRNA's are incorporated into avian sarcoma virus particles. Supporting evidence was obtained in an in vitro binding assay between purified reverse transcriptase and unfractionated cellular tRNA's. However, the subset of tRNA's which associated with the genome in the 70S complex was determined primarily by the viral RNA. In the absence of DNA polymerase, the 70S RNA complex in mature virus particles contained the normal complement of associated tRNA's with the exception of tRNATrp, the primer for RNA-directed DNA synthesis.

Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus.

We have previously described a nonconditional mutant of avian sarcoma virus (SE21Q1b) which fails to package viral RNA (Gallis et al., Virology 94:146-161, 1979; Linial et al., Cell 15:1371-1381, 1978). Quail cells transformed by SE21Q1b contain normal amounts of intracellular viral mRNA's for src, env, and gag-pol and release particles with the density of normal virus containing a typical complement of virion proteins, including reverse transcriptase. These virions are noninfectious for both chicken and quail cells and contain primarily cellular rather than viral RNA. Analysis by gel electrophoresis of the cellular DNA of quail cells transformed by SE21Q1b after restriction endonuclease digestion indicated the presence of a single provirus. The provirus was located at one site in the genome of the host cell and was flanked by the characteristic terminally repeated sequences derived from the 3' and 5' ends of viral RNA. The only defect detected in the SE21Q1b provirus was a deletion of ca. 150 base pairs of DNA somewhere between 300 and 600 bases from the left (gag-pol) end of the provirus. Analyses of the proviral DNA of cells transformed by wild-type recombinants between SE21Q1b and leukosis viruses reveal that the recombinants no longer contain this deletion. The deletion, therefore, defines a region on the viral RNA which is required for correct packaging of the virion RNA.