Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA.     

RNA metabolism of murine leukemia virus. III. Identification and quantitation of endogenous virus-specific mRNA in the uninfected BALB/c cell line JLS-V9.

mRNA containing type C endogenous virus-specific sequences was indentified in JLS-V9 cells (an uninfected BALB/c-derived cell line) by annealing extracted RNA with 3H-labeled virus-specific DNA. The criterion for virus-specific RNA being mRNA was that it co-sedimented with polyribosomes in a sucrose gradient and that it changed to lower sedimentation value if polyribosomes were disagregated prior to centrifugation. It was not possible to identify virus-specific mRNA in unfractionated cytoplasm from JLS-V9 cells since large amounts of virus-specific ribonucleoprotein which was not mRNA had sedimentation values similar to polyribosomes and obscured the analysis. Virus-specific mRNA could be readily identified in polyribosomes which had been purified through a step gradient of 1 and 2 M sucrose, and consisted of two species with sedimentation values of 38S and 27S. The amount of virus-specific RNA in different JLS-V9 cell fractions was quantitated in comparison to cell fractions obtained from M-MuLV clone no. 1 cells (a line of NIH 3T3 cells producing Moloney murine leukemia virus). Approximately 40% of the total virus-specific mRNA was recovered in the purified polyribosomes in M-MuLV no. 1 cells. The amount of virus-specific RNA on polyribosomes appeared to be quite similar for JLS-V9 cells and M-MuLV clone no.1 cells .In contrast, the level of virus-specific protein in JLS-V9 cells (as monitored by radioimmunoassay of the internal structural protein p30) was less than 2% the level in the M-MuLV clone no. 1 cells.     

Size analysis and relationship of murine leukemia virus-specific mRNA''s: evidence for transposition of sequences during synthesis and processing of subgenomic mRNA.

Virus-specific mRNA from purified polyribosomes of mouse cells infected with Moloney murine leukemia virus (M-MuLV) was analyzed by electrophoresis in agarose gels, followed by hybridization of gel slices with M-MuLV-specific complementary DNA (cDNA). The size resolution of the gels was better than that of sucrose gradients used in previous analyses, and two virus-specific mRNA's of 38S and 24S were detected. The 24S virus-specific mRNA is predominantly derived from the 3' half of the M-MuLV genome, since cDNAgag(pol) (complementary to the 5' half of the M-MuLV genome) could not efficiently anneal with this mRNA. However, sequences complementary to cDNA synthesized from the extreme 5' end of M-MuLV 38S RNA (cDNA 5') are present in the 24S virus-specific mRNA, since cDNA 5' (130 nucleotides) efficiently annealed with this mRNA. The annealing of cDNA 5' was not due to repetition of 5' terminal nucleotide sequences at the 3' end of M-MuLV 38S RNA, since smaller cDNA 5' molecules (60 to 70 nucleotides), which likely lack the terminal repetition, also efficiently annealed with the 24S mRNA. The sequences in 24S virus-specific mRNA recognized by cDNA 5' are not present in 3' fragments of virion RNA that are the same length. Therefore, it appears that RNA sequences from the extreme 5' end of the M-MuLV genome may be transposed to sequences from the 3' half of the M-MuLV 38S RNA during synthesis and processing of the 24S virus-specific mRNA. These results may indicate a phenomenon similar to the RNA splicing processes that occur during synthesis of adenovirus and papovavirus mRNA's.     

Analysis of cytoplasmic RNA and polyribosmomes from feline leukemia virus-infected cells.

Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA.     

The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses

Ribonucleic acids extracted from polyribosomes of cells replicating murine sarcoma-leukemia viruses (M-MSV(MLV)) were resolved by electrophoresis on 2.5% polyacrylamide gels. Virus-specific RNA was detected by hybridization of RNA in the gel fractions with the ³H-DNA product of the viral RNA-directed DNA polymerase. The postmicrosomal supernatant and the free polyribosomes contained one peak of virus-specific RNA with a molecular weight of about 2.9 × 10⁶ (35S). In contrast, the microsomes and the membrane-bound polyribosomes contained two peaks of virus-specific RNA in approximately equal amounts with molecular weights of 2.9 × 10⁶ (35S) and 1.5 × 10⁶ (approximately 20S). The high molecular weight viral RNA species might serve as polycistronic mRNA for the synthesis of large polypeptides that are cleaved to form the smaller viral proteins.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs results in the death of the animals within 12 to 15 days. Death is preceded by the simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellular virus particles, and (iii) a particle-associated RNA-directed DNA polymerase. This enzyme activity has a cation preference identical to that of the type B bromodeoxyuridine-induced guinea pig virus, i.e., an Mg2+ optimum at 20 mM and no activity using Mn2+. Competitive molecular hybridization studies also revealed that the plasma of leukemic guinea pigs contained approximately 2 X 10(9) genome equivalents per ml of an RNA that is homologous to the RNA of the bromodeoxyuridine-induced guinea pig virus. Morphological observations indicate that most, but not all, of the extracellular particles observed in leukemia plasma are derived from the intracisternal particles seen in the L2C tumor cells. The possibilities that either two viral populations are present or that the in vivo morphogenesis of the type B bromodexoyuridine-inducible guinea pig virus is markedly different from its in vitro morphogenesis are discussed.     

Mason-Pfizer virus RNA genome: relationship to the RNA of morphologically similar isolates and other oncornaviruses.

The 60-70S RNA of Mason-Pfizer virus (MPV) was iodinated in vitro and used in both direct and competitive molecular hybridization studies. MPV proviral sequences are present at a frequency of approximately one to two copies per haploid genome in the DNA of experimentally infected human cells. By nucleic acid competition hybridization, MPV RNA was found to be indistinguishable from the RNA of a virus (X381) isolated from a rhesus mammary gland and from RNA isolated from the cytoplasm of AO cells (Parks et al., 1973) and HeLa cells (Gelderblom et al., 1974), both previously reported to produce MPV-related particles. No homology was observed, however, between MPV RNA and the RNA, or the DNA, from two clones of HeLa cells obtained from the American Type Culture Collection. Hybridization of MPV 60-70S RNA to the DNA of normal tissues of humans and to the DNA of 11 other species revealed that MPV is not an endogenous virus of any of these species. Competition hybridization revealed no detectable sequence homology between the RNA of MPV and the RNAs of simian sarcoma virus, murine mammary tumor virus, murine leukemia virus, BUdR-induced guinea pig virus, or avian myeloblastosis virus. These nucleic acid studies substantiate previous ultrastructural and immunological findings that MPV and morphologically similar isolates constitute a distinct group of oncornavirus.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit

Different fractions of cellular RNA from erythroid enriched bone marrow cells of the rabbit, extracted by the temperature fractionation method, were investigated by hybridization to globin cDNA. 97.4% of all globin sequences were found in the 4 degrees C franction (cytoplasmic RNA) 0.11% are in the 40 degrees / 50 degrees C fraction and 2.47% in the 65 degrees C and 85 degrees C franctions (pre-mRNA). This shows a substantial purification of the pre-mRNA fractions from cytoplasmic mRNA. 33% of the globin sequences in the 65 degrees C and 85 degrees C fractions are polyadenylated. The poly(A)+-RNA from the 65 degrees C and 85 degrees C fractions separated in a formamide sucrose gradient showed a clear hybridization to globin cDNA in the region between 9S and 28S and around 4S. In a control experiment in which RNA from baby hamster kidney cells (BHK) was mixed with globin mRNA and separated in the same manner hybridization was observed at the 9S position of the gradient only.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

RD 114 Virus-Specific Sequences in Feline Cellular RNA: Detection and Characterization

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.     

Ribonucleic Acid Synthesis in Cells Infected with Influenza Virus

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34S, 18S, and 11S. These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34S RNA species is an aggregate of 18S and 14S RNA species. Both 18S and 11S RNA species are relatively heterogenous compared to 18S ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.     

RNA metabolism of murine leukemia virus II. Endogenous virus-specific RNA in the uninfected BALB/c cell line JLS-V9.

Type C virus-specific RNA sequences of BALB/c endogenous virus were detected in JLS-V9 cells (an uninfected BALB/c derived line) by annealing cell RNA with 3-H-labeled virus-specific DNA. Endogenous viruses used in preparing the 3-H-labeled DNA (mostly xenotropic) was prepared from JLS-V9 cells induced to produce virus with iododeoxyuridine. In whole-cell extracts, two virus-specific RNA species, 38S and 27S, were detected. No 60 to 70S virus-specific RNA was found. The same two species of virus-specific RNA were observed in isolated cytoplasmic RNA and in cytoplasmic RNA selected for polyadenylic acid-containing species by binding and elution from oligo(dT) cellulose. Very little, if any, of the virus-specific RNA was active as messenger RNA on polyribosomes. No virus-specific RNA transcribed from genes coding for the BALB/c endogenous N-tropic virus was detected, since 3-H-labeled DNA prepared from endogenous N-tropic virus did not hybridize measurably with JLS-V9 RNA.     

5''-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.