Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B(77)), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB(77)), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Molecular basis of retrovirus adaptation: nucleotide sequence of Rous sarcoma virus adapted to duck cells

The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.     

Infectivity of Proviral DNA from Avian Sarcoma Virus-Transformed Mammalian Cells

The number of Rous viral genomes in the cellular DNA from two subclones (RS2/3, RS2/6) derived from the same clone of hamster BHK-21 cells transformed by Rous sarcoma virus was determined by hybridization with viral complementary DNA made in vitro, and the capacity of the cellular DNA to infect (transfect) chicken embryo fibroblasts was compared before and after shearing this DNA to about the size of the provirus (6 x 10(6) to 7 x 10(6) daltons). The two subclones differed widely both in their capacity to give rise to virus (inducibility) after fusion with chicken embryo fibroblasts and in level of expression of viral proteins. It was shown that cells of both subclones contain a single copy of Rous DNA and yield infectious DNA. However, whereas transfection of chicken embryo fibroblasts was successful with both unsheared (>/=18 x 10(6) daltons) and sheared DNA from the most inducible subclone (RS2/3 subclone), which also expresses viral proteins to an appreciable amount, transfection with DNA from the least inducible subclone (RS2/6 subclone), in which viral proteins are not expressed, succeeded only with sheared DNA. It was then about as successful as with sheared or unsheared RS2/3 DNA. The lack of infectivity of unsheared RS2/6 DNA may be explained by the hypothesis proposed by Cooper and Temin (G. M. Cooper and H. T. Temin, J. Virol. 17:422-430, 1976) to explain the lack of infectivity of DNA from certain chicken cells producing spontaneously low amounts of RAV-0 and resistant to exogenous RAV-0 infection, that is, that the viral genome (proviral DNA) is linked to a cis-acting control element which blocks its expression. This linkage might originate, in RS2/6 cells, from translocation of cellular DNA containing the single proviral copy.     

Independent expression of avian sarcoma virus in doubly infected chicken embryo fibroblasts.

Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus.     

Immunological and structural properties of Rous sarcoma virus-transformed and tumor cells]

We compared the capacity of both normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells as well as Rous sarcoma (RS) tumor cells to serve as targets in anti-tumor immunity assays. These studies showed that sera from tumor-bearing donors were able to stain transformed CEF more efficiently than RS cells as detected by immunofluorescence. In contrast, antiserum against the major viral glycoprotein, gp 85, stained a higher percentage of RS than transformed cells. Normal CEF cells, which served as controls, were essentially non-reactive with the immune system as judged by this type of assay. We observed that RS cells are considerably larger and contain far higher levels of protein than either normal or transformed CEF. Scanning electron microscopy revealed both the RS cells and transformed CEF to be rich in surface ruffles, blebs and microvilli as distinguished from the flattened, fusiform appearance of normal CEF cells.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Low resistance junctions between normal and between virus transformed fibroblasts in tissue culture

The occurrence of low resistance junctions between normal chick embryo fibroblasts and between fibroblasts transformed with Rous sarcoma virus in tissue culture was studied with intracellular microelectrodes. The results showed that these junctions were present between normal chick fibroblasts in proliferating cultures as well as between cells in ‘density dependent inhibited cultures’. Mechanical injury to a fibroblast within a small group of coupled cells caused the injured fibroblast to uncouple immediately from its neighboring cells without interrupting coupling between the healthy uninjured cells. In the case of fibroblasts transformed with a Rous sarcoma virus, the results further showed that low resistance junctions were present when the transformation appeared in the infected cells and remained present thereafter.     

Integration of Rous sarcoma virus DNA into chicken embryo fibroblasts: no preferred proviral acceptor site in the DNA of clones of singly infected transformed chicken cells.

We analyzed retroviral integration into a host genome by using avian sarcoma virus infection of natural target cells under conditions where secondary integration via virus spread was inhibited. This was accomplished by using the noninfectious pol- env- alpha variant of the Bryan high-titer strain of Rous sarcoma virus. A total of 12 independent Bryan high-titer Rous sarcoma virus-transformed chicken embryo fibroblast clones were obtained and mapped by using restriction endonucleases. Provirus-cell junction fragments were identified with appropriate hybridization probes. We found that expression of the viral genes could occur after proviral integration at many sites on the chicken genome and that there was no apparent preference for specific integration sites.     

Virus Recovery in Chicken Cells Tested with Rous Sarcoma Cell DNA

DNA from non-virus-producing RSV transformed mammalian cells converts chicken fibroblasts into Rous sarcoma cells producing infectious RSV particles. The recovered virus is the same biologically and antigenically as the virus which originally transformed the mammalian cells.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.