Anti-Rous sarcoma response of major histocompatibility (B) complex haplotypes B23, B24 and B30

Responses to Rous sarcoma virus (RSV) induced tumours were studied in UNH 105, a non-inbred line of New Hampshire chickens. Six single male matings encompassing a total of 50 dams produced 345 progeny which segregated for B complex genotypes B23/B23, B23/B24, B23/B30, B24/B24, B24/B30 and B30/B30. Six-week-old chicks were wingweb inoculated with a pseudotype of Bryan high titre Rous sarcoma virus, BH RSV (RAV-1). Tumours were scored for size six times over a 10-week period post-inoculation. Each chick was assigned a tumour profile index (TPI) as an indicator of immunological response. The number of days to death (DTD) was recorded for 148 chicks with terminal tumours. Genotypes B23/B23, B23/B24 and B23/B30, with TPIs of 1.8, 1.7 and 2.0 respectively, did not differ significantly from each other, suggesting dominance of response of B23 over B24 and B30 haplotypes. B24/B30 chicks with the highest TPI (3.4) and shortest DTD (34.6) were significantly different from B30/B30 (2.8; 41.6) but not from B24/B24 (3.1; 34.9) suggesting dominance of response of the B24 haplotype over B30 in the absence of B23.     

Letter: Evidence for non-repetitive subunits in the genome of Rous sarcoma virus

The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 10⁶ daltons, a value close to its physically-determined molecular weight of about 10⁷. Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded.     

Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library

The chicken major histocompatibility complex (MHC-B locus) has a strong association with resistance and susceptibility to numerous diseases. We have found a B haplotype designated WLA that associated with the regression of tumours caused by Rous sarcoma virus J strain (RSV-J). Haplotype WLA was identical to the regressive B6 haplotype when partial genotyping was performed (Poultry Science, 89, 2010, 651). We then constructed a bacterial artificial chromosome (BAC) library from a WLA homozygote chicken to evaluate the structure of this regression haplotype and compared it to those of the B6 haplotype. Comparison between WLA and B6 above 59 kb within the 167 kb, including 14 genes from BG1 to BF2, revealed 75 SNPs and 14 indels. However, several genes were identical between WLA and B6, including the BF1 and BF2 genes, which encode a class I molecule previously suggested to be related to the regression phenotype. The BLB2 gene encoding the MHC class II beta chain showed the greatest diversity, with 19 non-synonymous SNPs. A comparison of WLA and B6 haplotpyes that are associated with tumour regression and RIRa and B24 haplotypes associated with tumour progression suggests that DMA1, DMA2, BRD2, TAPBP and BLB2 genes are not involved in the intensity of RSV J tumour regression.     

Localization of N6-methyladenosine in the Rous sarcoma virus genome.

The 10,000-nucleotide RNA genome of the Prague strain, subgroup B (PR-B) of Rous sarcoma virus, was found to contain 11.6 ± 0.5 residues of m⁶Ap by quantitative analysis of ³²P-labeled virion RNA after complete RNAase digestion. Approximately ten of the m⁶Ap residues are located, without obvious clustering, in that region of the genome between 500 and 4000 nucleotides from the 3′ poly(A) end. The src gene, which is required for transformation, and part of the env gene, which codes for the major viral envelope glycoprotein, have previously been mapped in this region of the viral genome. A transformation-defective deletion mutant of PR-B Rous sarcoma virus, which lacks the src gene, has 7.0 ± 0.2 m⁶Ap residues per RNA subunit. This supports our mapping of a portion of the m⁶A residues in src and suggests that this methylation is specific to certain regions of the genome. The possible significance of this result for Rous sarcoma virus RNA processing and translation is discussed.     

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.     

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453–462, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of Rous sarcoma virus capsid protein variants assembled on lipid monolayers

During assembly and morphogenesis of Rous sarcoma virus (RSV), proteolytic processing of the structural precursor (Pr76Gag) protein generates three capsid (CA) protein variants, CA476, CA479, and CA488. The proteins share identical N-terminal domains (NTDs), but are truncated at residues corresponding to gag codons 476, 479, and 488 in their CA C-terminal domains (CTDs). To characterize oligomeric forms of the RSV CA variants, we examined 2D crystals of the capsid proteins, assembled on lipid monolayers. Using electron microscopy and image analysis approaches, the CA proteins were observed to organize in hexagonal (p6) arrangements, where rings of membrane-proximal NTD hexamers were spaced at 95 A intervals. Differences between the oligomeric structures of the CA variants were most evident in membrane-distal regions, where apparent CTDs interconnect hexamer rings. In this region, CA488 connections were observed readily, while CA476 and CA479 contacts were resolved poorly, suggesting that in vivo processing of CA488 to the shorter forms may permit virions to adopt a dissembly-competent conformation. In addition to crystalline arrays, the CA479 and CA488 proteins formed small spherical particles with diameters of 165-175 A. The spheres appear to be arranged from hexamer or hexamer plus pentamer ring subunits that are related to the 2D crystal forms. Our results implicate RSV CA hexamer rings as basic elements in the assembly of RSV virus cores.     

Effects of therapeutic treatment with the MER tubercle bacillus fraction on Rous sarcomas of chickens induced by the Rous sarcoma virus

Summary Multiple concurrent injections of MER, to give a total dose of 0.5 mg, at the site of palpable Rous sarcomas developing in outbred chickens following infection with the Rous sarcoma virus effected seemingly permanent regression of the neoplasms in 25 of 76 subjects. In contrast, administration of the same amount of the agent to the tumour area in a single injection, in a small volume, was effective in only 2 of 54 birds.     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.     

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.     

Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70–160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0–1.4 μg/10⁷ sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25–30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60^src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed. © 1996 Wiley-Liss, Inc.     

Major histocompatibility (B) complex and sex effects on the phytohaemagglutinin wattle response

The influence of the major histocompatibility (B) complex and sex on the phytohaemagglutinin (PHA) wattle response was studied in 136 segregants (B2/B2, B2/B5 and B5/B5) of a fourth generation cross between inbred lines 6(1) and 15(1). At 6 weeks of age, chickens were injected with 100 micrograms purified PHA-P. Wattle thickness measurements were taken 4, 24, 48, 72 and 96 h after injection. Analysis of variance showed that 4 h after injection, males had a significantly higher response than females but the sex-genotype interaction was also significant. Females had higher responses than males 24 and 48 h after injection as a consequence of more rapid development and earlier resolution of the reaction in males. B2/B2 chickens had significantly lower responses than B5/B5 chickens 72 and 96 h after injection, signifying a faster late resolution phase in the B2/B2 genotype. The developmental and early resolution phases of the PHA wattle response were influenced by sex while the late resolution phase was influenced by B genotype.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Interaction of the B locus and the GVHR-selected lines in the graft-versus-host reaction in chickens

Genetic differences affecting the degree of splenomegaly in the graft-versus-host reaction (GVHR) of chickens were studied. Two B genotypes, B⁹B⁹ and B¹¹B¹¹, and two GVHR-selected lines, H and L, were examined. The degree of splenomegaly of B⁹ B⁹→ B¹¹B¹¹ was significantly higher than that of B¹¹B¹¹→ B⁹B⁹ for all line combinations. In contrast, the inoculation of H into L gave consistently higher splenomegaly than that of L into H. This suggested that the effects of B locus were higher in hosts than in donors, while those of the GVHR-selected lines were higher in donors than in hosts.
The analysis of variance revealed that both the differences between the reciprocal combination of B genotypes and between the GVHR line combinations were statistically highly significant. Furthermore, the interaction of B genotypes and GVHR lines was also highly significant.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.     

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1-¹⁴C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1-¹⁴C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [³⁵S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.