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

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

Electron microscopy of viral RNA: avian tumor virus RNA.

We have investigated by electron microscopy the structure of native, partially denatured, and heated and quick-cooled RNA from avian myeloblastosis virus (AMV) and Schmidt-Ruppin Rous sarcoma virus (SR-RSV). Native 60–70 S RNA has a highly folded structure, while partially denatured 60–70 S RNA is more extended but retains much secondary and tertiary structure and has multiple free ends. 60–70 S RNA that has been heated and quick-cooled appears smaller than unheated RNA and retains some secondary structure when prepared for electron microscopy under nondenaturing conditions. The appearance of 60–70 S RNA before and after heating indicates a dissociation of the 60–70 S RNA on heating rather than a change in conformation of the RNA. Molecular weight of large subunit RNA has been determined by electron microscopy of formaldehyde-formamide treated RNA. The largest RNA subunit of AMV has a molecular weight of 2.9 × 10⁶. The largest subunit of the transforming virus SR-RSV is 20% larger with a molecular weight of 3.5 × 10⁶.     

Ultraviolet inactivation of influenza virus RNA in vitro and vivo.

The uv inactivation of influenza virus RNA within the infected cell indicated that each segment was being transcribed individually. That is, each segment carried its own promoter region. Similar results were obtained when RNA synthesis by uv-irradiated virus was tested in vitro.     

Host antigens on avian oncoviruses: evidence for virus envelope antigens related to specific chicken erythrocyte membrane antigens

Avian sarcoma viruses (ASV) of subgroups A to D, produced by chick embryo fibroblasts (CEF), are inactivated to a high degree by rabbit antisera to the membrane antigens of adult chicken and chick embryo erythrocytes, notably by antisera to an antigen of embryo erythrocytes, which is lost by adult erythrocytes and to another antigen specific to the latter erythrocytes. Contrary to virus inactivation by anti-CEF serum reported earlier, virus inactivation by the antisera to these two age-specific antigens does not require complement and is not paralleled by virolysis but by aggregation of virions. The two antigens related, or identical, to the age-specific erythrocyte membrane antigens thus shown to be present on the virus envelope do not pre-exist, or pre-exist only in a low amount, on the CEF membrane, since the virus-inactivating capacity of their antisera is not removed by absorption with CEF. Their appearance on the virus does not depend on cell transformation but only on infection, since both antigens are found on a ts ASV mutant produced at restrictive temperature by untransformed CEF and the virus-inactivating capacity of their antisera is removed by absorption with CEF infected with Rous-associated virus (RAV-1). These findings suggest that infection of CEF by avian oncoviruses may elicit the appearance, or enhance the expression at the cell surface of antigens characteristic of another cell type which may contribute to the formation of specific virus budding sites.     

A comparison of the high molecular weight RNAs of visna virus and Rous sarcoma virus

Visna virus RNA consists of a high molecular weight and low molecular weight species. The size, subunit composition, complexity, and secondary structure of high molecular weight RNA have been studied and compared with Rous sarcoma virus RNA. Visna high molecular weight RNA cosediments in 0.1 M sodium chloride wiih the 70S UNA of Rous sarcoma virus consistent with a molecular weight of 10–12 × 10 daltons. On dissociation with heat, subunit structures of 2.8 × 10⁶ daltons are released. Both the size and heterogeneity of the subunit RNA are identical with the subunit RNA of transforming Schmidt-Ruppin Rous sarcoma virus. Comparison of the complexity of visna RNA with poliovirus RNA indicate unique nucleotide sequences of 7–10 × 10⁶ daltons, in accord with the physical data. The evidence also indicates that there is lack of significant reiteration of nucleotide sequences in the visna RNA. Major differences in the secondary structure of visna and Rous sarcoma virus RNA were observed. Both RNAs possess an exceptional degree of secondary structure when assayed by chromatography on cellulose at various temperatures. Reduction of ionic srength markedly decreases the secondary structure of visna RNA relative to Rous sarcoma virus RNA and leads to anomalous migration in polyacrylamide gels. The differences in secondary structure and in the low molecular weight RNA species associated with 70S RNAs of these viruses indirectly implicate the 4 and 5S RNA in the maintenance of secondary structure of 70S RNA of Rous sarcoma virus.     

RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus

Gel electrophoresis of the dissociated 60–70 S RNA prepared from cloned avian sarcoma viruses showed a single major peak corresponding to size class a. Class b RNA was not detectable in these preparations. Class a RNA derived from the Schmidt-Ruppin strain of Rous sarcoma virus had a slightly but reproducibly lower electrophoretic mobility than class a RNA of Prague strain Rous sarcoma virus. The presence of avian tumor virus group specific (gs) antigens in chicken cells before infection did not detectably influence the electropherograms of the dissociated avian sarcoma virus RNA: cloned sarcoma virus from gs positive and gs negative cells showed identical gel patterns. However, two avian sarcoma virus clones derived from a clonal colony of transformed cells contained a new RNA component which migrated slower than class a RNA. This unusual component was found consistently in the original clonal cultures but was not seen if new cells were infected with the same virus, suggesting a cell mediated modification of viral RNA.Additional evidence was obtained for the absence of class a RNA from avian tumor viruses which cannot form foci in fibroblast cultures. Several leukosis and transformation defective viruses which had not been analyzed previously showed class b RNA only. No significant differences between the class b RNA preparations obtained from several independently isolated transformation defective viruses were detected.     

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.     

Intracellular and virion 35 S RNA species of murine sarcoma and leukemia viruses

RNA molecules from the following sources were treated with dimethyl sulfoxide to dissociate noncovalent aggregates and resolved by electrophoresis on polyacrylamide gels: (1) transformed MSV(MLV)-producing Balb/3T3 cells (MSV-39, clone 24), (2) transformed MSV(MLV)-producing rat cells (78A1), (3) MSV transformed, non virus-producing hamster cells (HT-1), (4) MLV-producing Balb/3T3 cells, (5) MLV-producing NIH/3T3 cells (6) 60–70 S RNA from MSV(MLV) virions, and (7) 60–70 S RNA from MLV virions. Virus-specific RNA was detected by hybridization of RNA in gel fractions with the ³H-DNA product of the MSV(MLV) RNA-directed DNA polymerase. Two well defined viral RNA species with sedimentation coefficients of 35 S and 20 S, but none of intermediate size, were detected in both MSV(MLV) producing cell lines. The non virus-producing HT-1 cell line contained a viral RNA species slightly smaller than 35 S, about 33 S, but no detectable 20 S virus-specific RNA. These results with 78A1 and HT-1 cells agree with previous conclusions based on rate-zonal centrifugation in sucrose density gradients (Tsuchida et al., 1972). The two MLV-producing mouse cell lines contained a well defined 35 S RNA peak, a somewhat less pronounced 20 S RNA peak, and heterogeneous RNA species of smaller molecular weights. Although both 35 S and 20 S virus RNA species were detected in cells replicating murine oncornaviruses, labeled 60–70 S RNA isolated from MSV(MLV) virions (consisting mainly of MLV) and MLV virions, and dissociated with dimethyl sulfoxide or by heat, consisted of 35 S RNA and about 10–15% 7 S RNA plus 4–5 RNA, but no detectable 20 S RNA. Hybridization-competition experiments using MSV(MLV) 60–70 S ³H-RNA (consisting mainly of MLV RNA) and saturating amounts of the unlabeled DNA product of the MSV(MLV) RNA-directed DNA polymerase showed that 35 S RNA from cells replicating MSV(MLV) shares virtually all of its nucleotide sequences with MSV(MLV) 70 S RNA; these data suggest that intracellular 35 S RNA species are the major precursors to virion 70 S RNA. In contrast, 33 S RNA from HT-1 cells shares only about 50% of its sequences with MSV(MLV) 70 S RNA indicating that only part of the sequences of MSV(MLV) 60–70 S RNA are integrated and/or transcribed in this non virus-producing MSV transformed cell.     

The RNA of human coronavirus OC-43.

A homogeneous RNA complex with a sedimentation coefficient of 70 S and an apparent molecular weight of approximately 6.1 × 10⁶ was released from purified ³²P-labeled, mouse-brain-derived OC-43 virus after treatment with 1% sodium dodecyl sulfate (SDS) for 15 min at 23°. The complex was highly susceptible to heat, releasing 4 S RNA fragments at 37° and breaking down to fragments of 4–70 S at 60°; it was also degraded by centrifugation through dimethyl sulfoxide gradients. Unlike tobacco mosaic virus or Rous sarcoma virus RNA, OC-43 RNA prepared by extraction with phenol-SDS or phenol-chloroform degraded into a range of fragments with coefficients of 15–55 S; 4 S RNA was also present as a minor component. This suggests that (a) extensive nicking of a large RNA molecule has occurred during viral growth, due to ribonucleases which are inactivated during phenol extractions; (b) heterogeneity for OC-43 RNA is not due to internal ribonuclease activity and fragments are held together by noncovalent linkages much weaker than those present in the 70 S retroviral RNA complex, or by small proteins; or, most probably, (c) a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products.     

Presence of two “8 S” RNA components in mouse sarcoma virus (Moloney)

RNA was prepared from the murine leukemia virus pseudotype of mouse sarcoma virus. On polyacrylamide gels, 70 S single-stranded RNA was detected as well as smaller RNA species of 28 S, 18 S, and 4 S. In addition, two components of 8 S were discovered. These 8 S RNA molecules may correspond to the 7 S RNA of Rous sarcoma virus and to the 9 S of avian myeloblastosis virus.     

The relationship of visna, maedi and RNA tumor viruses as studied by molecular hybridization

The DNA product of the endogenous visna virus RNA-directed polymerase reaction annealed to maedi virus 60–70S RNA. Similarly, the DNA product of the endogenous maedi virus polymerase reaction hybridized to visna virus 60–70S RNA. The DNA products of endogenous visna and maedi virus polymerase reactions failed to anneal significantly either to Rauscher murine leukemia or mouse mammary tumor virus 60–70S RNAs.     

Comparison of oligonucleotides produced by RNase T1 digestion of 7 S RNA from avian and murine oncornaviruses and from uninfected cells

The 7 S RNA molecules isolated from Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), mouse sarcoma virus (MSV), and L cell virion (LCV) were treated with RNase T1, and the oligonucleotides produced were fractionated by two-dimensional electrophoresis. The 7 S RNAs from RSV and AMV yield identical fingerprints. Thirty-five oligonucleotides are observed in yields greater than 0.5 mole per mole RNA, and the molecules consist of about 280 nucleotides. LCV and MSV 7 S RNAs show a difference of two oligonucleotides of a total of 37. Comparison of avian and murine 7 S RNAs also shows a large number of similarities, including the 3′ terminus (U₄C₂)X_OH. The results indicate that large portions of the sequence of 7 S oncornavirus RNAs have been conserved among viruses that replicate in different hosts. Analysis of 7 S RNA from uninfected chick fibroblasts demonstrates that they contain the same molecule as that found in AMV and RSV.     

The use of guanidine-HCl for the isolation of both RNA and protein from RNA tumour viruses.

The RNA components of two C-type RNA viruses, avian myeloblastosis virus and Friend leukaemia virus, have been isolated by treatment of the viruses with 6 M-guanidine-HCl and precipitation with ethanol. The virus proteins were recovered by lyophilization of the guanidine-HCl-ethanol supernatant after thorough dialysis against 0.5 mM-dithiothreitol. This simple method yielded RNA of similar quality to the phenol and sodium dodecyl sulphate (SDS) extraction methods, and the same amount of 60-70S RNA, although a fraction of the smaller (4S) species remained in the protein fraction. The sedimentation patterns of heat-denatured RNA extracted by either method were similar. Electrophoretic analyses of the extracted proteins in polyacrylamide gel gradients containing SDS gave patterns that were very similar to those obtained by direct analysis of SDS disrupted viruses.     

The DNA of murine sarcoma-leukemia virus

A DNA wih a sedimentation coefficient of 7 S (ρc_s2so₄: = 1.423) was isolated from DNase- and RNase-treated purified virions of each of three different oncogenic RNA viruses: murine sarcoma virus, Rauscher murine leukemia virus, and avian myeloblastosis virus. The naturally occurring DNA of murine sarcoma virus constituted about 2.5% of the total viral nucleic acid and resolved into two components (ρ = 1.698 and ρ = 1.678) when centrifuged to equilibrium in a CsCl density gradient. The viral DNA was complementary to the 18 S viral RNA subunit, but not to the 37 S or 4 S subunits.     

Viral RNA of murine sarcoma virus produced by a hamster-mouse somatic cell hybrid

The viral RNA genome of murine sarcoma virus synthesized by a hybrid cell, THR-23, which was isolated by Sendai virus fusion of a hamster cell (HT-1) carrying the defective Moloney sarcoma genome and Swiss mouse cells shedding Rauscher leukemia virus was characterized. The THR-23 clone produces a 500-fold excess of sarcoma over leukemia virus. The high molecular weight (HMW) RNA from the virion of THR-23 gave a single peak in sucrose gradients with a sedimentation coefficient of 60 S, smaller than that of Rauscher murine leukemia virus (70 S). The 60 S HMW RNA of THR-23 virus dissociates to a 30 S subunit RNA after heat denaturation. The size is different from 35 S subunit RNA of 70 S RLV RNA. 30 S intracellular virus-specific subunit RNA was detected in HT-1 and THR-23 cells by hybridization with DNA product made in vitro from Moloney murine sarcoma and leukemia virus. These results suggest that the defective genome (30 S RNA) of the HT-1 cell is synthesized in the THR-23 hybrid cell, aggregated to 60 S viral HMW RNA and incorporated into the virion. In contrast to the avian system, the murine sarcoma genome is smaller by sedimentation than the murine leukemia genome.     

Homologies among the nucleotide sequences of the genomes of C-type viruses

DNA was synthesized with detergent-disrupted virions of several C-type viruses and used to measure the extent of homology among the genomes of these viruses by molecular hybridization. The DNA was reacted with viral RNA under conditions which permit saturation of most if not all complementary nucleotide sequences in the RNA. This technique provides a quantitative estimate of the extent of homology among viral RNAs and is superior to current procedures that measure the fraction of DNA hybridized to an excess of viral RNA. The genomes of RD-114 and Crandell virus are at least 85% related, whereas there is no detectable homology among the genomes of RD-114 virus, feline sarcoma-leukemia viruses, murine leukemia virus, avian sarcoma virus, and visna virus. The genome of feline sarcoma-leukemia viruses, like that of RD-114 virus, is at least partly homologous to DNA from normal cats, suggesting that normal cats harbor endogenous genes coding for components of at least two classes of C-type viruses.