Complexity and polyadenylic acid content of visna virus 60-70S RNA.

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.     

Base sequence differences between the RNA components of Harvey sarcoma virus.

The 50 to 70S RNA of the Harvey sarcoma-Moloney leukemia virus (MLV) complex consists of 30 to 40S RNA subunits of two different size classes and contains sequences homologous to Moloney mouse leukemia virus and to information contained in a C-type rat virus, termed NRK virus. We have isolated by preparative gel electrophoresis the large (component 1) and the small (component 2) 30 to 40S RNA species from the Harvey sarcoma-MLV complex. Harvey RNA component 1 was completely complementary to DNA transcribed from MLV RNA and showed no homology to DNA transcribed from NRK virus when annealed under conditions of DNA excess. Harvey RNA component 2 was about 65% complementary to MLV DNA and about 33% complementary to NRK virus DNA. Approximately 60 to 80% of the MLV-specific sequences in RNA component 2 is either a distinct molecular species or is part of a hydrid molecular including NRK virus- and MLV-specific sequences. The rest of the MLV sequences in component 2 could be accounted for by degraded component 1 co-purifying with component 2. The possible role of these sequences in the ability of the virus to transform cells is discussed.     

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.     

Role of Subunits of 60 to 70S Avian Tumor Virus Ribonucleic Acid in Its Template Activity for the Viral Deoxyribonucleic Acid Polymerase

Heating the 60 to 70S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70S structure dissociates into 30 to 40S and several distinct small subunits with a T(m) of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (<10S) RSV RNA primer. Sixty percent of the high template activity of 60 to 70S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40S subunits isolated from 60 to 70S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (<10S) subunits also isolated from 60 to 70S RNA, the template activity of 30 to 40S RNA subunits was increased to the same level as that of reannealed total 60 to 70S RNA. It was concluded that neither the 30 to 40S subunits nor most of the 4S subunits of 60 to 70S RSV RNA contribute much as primers to the template activity of 60 to 70S RSV RNA. The predominant primer molecule appears to be a minor component of the <10S subunit fraction of 60 to 70S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70S RSV RNA is higher than that of the bulk of 60 to 70S RSV RNA-associated 4S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.     

Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs.

Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.     

Properties of feline leukemia virus. III. Analysis of the RNA.

The kinetics of virus labeling was used to study the maturation of viral RNA in the Rickard strain of feline leukemia virus. Viral RNA labeled over differing intervals was characterized by gel electrophoresis and velocity sedimentation in sucrose gradients made up in aqueous buffer and 99% dimethyl sulfoxide. Labeled virus was found within 30 min after adding radioactive uridine to the cells and production of labeled virus reached a maximum at 4 to 5 h after pulse labeling. Native RNA from feline leukemia virus resolved into three size classes when analyzed by electrophoresis on 2.0% polyacrylamide-0.5% agarose gels: a 6.2 x 10(6) to 7.1 x 10(6) mol wt (50 to 60S) class, an 8.7 x 10(4) mol wt (approximately 8S) class, and a 2.5 x 10(4) mol wt (4 to 5S) class. From two experiments during which RNA degradation appeared minimal, these made up to 57 to 76%, 2 to 5%, and 6 to 12%, respectively, of the total RNA. The 8S RNA in feline leukemia virus has not previously been reported. The 50 to 60S RNA from virus harvested after 4 h of labeling electrophoretically migrated faster and sedimented more slowly in sucrose gradients than did the same RNA species harvested after 20 h of labeling. This argues for an intravirion modification of the high-molecular-weight RNA. The large subunits of denatured viral RNA from both 4- and 20-h labeled-viral RNA electrophoretically migrated with an estimated molecular weight of 3.2 x 10(6) but sedimented with 28S ribosomal RNA (1.8 X 10(6) mol wt) when analyzed by velocity sedimentation through 99% dimethyl sulfoxide.     

Electrophoretic Characterization of Foot-and-Mouth Disease Virus-Specific Ribonucleic Acid

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.     

Nucleic Acid of Rubella Virus and Its Replication in Hamster Kidney Cells

Ribonucleic acid (RNA) has been isolated from partially purified rubella virus preparations and fractionated by rate zonal centrifugation in sucrose density gradients. The bulk of the RNA sedimented as a sharp band with a sedimentation coefficient of 38S. Rubella virus RNA appears to be single-stranded on the basis of its sensitivity to the degrading action of ribonuclease. Fractionation by precipitation with 1 m NaCl, followed by chromatography on cellulose columns, and by rate zonal centrifugation in sucrose density gradients of labeled RNA isolated from actinomycin D-treated and infected baby hamster kidney cells revealed the presence of the following virus-specific types of RNA: (i) single-stranded RNA with a heterogeneous sedimentation pattern, the 38S viral RNA becoming the predominant species only after long periods of labeling late after infection; (ii) double-stranded RNA with a sedimentation coefficient of 20S; (iii) RNA apparently composed of 20S double-stranded RNA and single-stranded branches. On the basis of their properties, the last two species were tentatively identified as the replicative form and the replicative intermediate of rubella virus RNA. Rubella virus RNA was infectious.     

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.     

Structure containing cellular mRNA in picornavirus infections

The fate of cellular mRNA upon infection of Krebs-2 ascites carcinoma cells with encephalomyocarditis (EMC) virus was investigated. The cell mRNA was discovered in a structure with a sedimentation coefficient of about 100S and a buoyant density of 1.50--1.519 g/cm3 during active virus-specific synthesis (3.0--4.0 hr post infection). The template activity of the 100S structure in a cell-free protein-synthesizing system and of mRNA isolated from it was studied and the nature of synthesized products was analyzed. It was shown that the 100S structure seems to be translationally inactive. On the contrary, the RNA isolated from its is functionally active.     

Different Classes of Ribonucleic Acid Isolated from Lymphocytic Choriomeningitis Virus

Purified preparations of lymphocytic choriomeningitis virus (LCM virus) contain three classes of RNA. The previously described 18s, 23s, 28s, and 31s RNAs, where the 23s and 31s RNAs are viral-specific, and the 18s and 28s RNAs probably are host RNAs incorporated in the virion. Now, 4s, 5s, and 5.5s RNAs can be isolated as well. Thus five RNAs which migrate by acrylamide gel electrophoresis as ribosome-derived RNA can be isolated from purified LCM virus. This observation further supports the reports that arenaviruses may contain ribosomes. The ribosome-derived RNA can be synthesized both before and after the virus infection. The viral 23s could be a hydrogen-bonded complex forming the 31s RNA, or it could be contained in defective interfering LCM virus particles; these possibilities are examined.     

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.     

Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA.

The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U) from RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase T1 contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'-terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 mug of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimetnally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus.     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Association of 4S Ribonucleic Acid with Oncornavirus Ribonucleic Acids

Oncornavirus 60 to 70S ribonucleic acids (RNA), such as those from avian myeloblastosis virus, Schmidt-Ruppin virus, or mouse sarcoma-mouse leukemia viruses, isolated by conventional techniques, contain 4S transferlike RNA molecules that are released upon dissociation of the 60 to 70S RNA with heat. The 4S RNA represents 2.5 to 3.0% of the RNA in the 65S aggregate or 4 to 5 molecules per molecule of 35S RNA formed.     

Characterization of Ribonucleic Acid from Visna Virus

A single-stranded ribonucleic acid(s) has been isolated from purified virions of visna virus. It consists of two major components, namely 63S and "4S," under the conditions employed for ribonucleic acid (RNA) extraction. The 63S component can be converted to subunits by heat and dimethylsulfoxide treatments. Analyses by base composition indicate that the "4S" RNA isolated from visna virus is not a random breakdown product of the 63S component as a result of extraction, nor is it randomly derived from cellular RNA.     

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.