Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.     

RNA-dependent RNA polymerase activity in coronavirus- infected cells

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.     

Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes

We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3.

The composition and structure of the mouse hepatitis virus (MHV)-specific RNA in actinomycin D-treated, infected L-2 cells were studied. SEven virus-specific RNA species with molecular weights of 0.6 X 10(6), 0.9 X 10(6), 1.2 X 10(6), 1.5 X 10(6), 3.0 X 10(6), 4.0 X 10(6), and 5.4 X 10(6) (equivalent to the viral genome) were detected. T1 oligonucleotide fingerprinting studies suggested that the sequences of each RNA species were totally included within the next large RNa species. The oligonucleotides of each RNA species were mapped on the 60S RNA genome of the virus. Each RNA species contained the oligonucleotides starting from the 3' end of the genome and extending continuously for various lengths in the 3' leads to 5' direction. All of the viral RNA species contained a polyadenylate stretch of 100 to 130 nucleotides and probably identical sequences immediately next to the polyadenylate. These data suggested that the virus-specific RNAs are mRNA's and have a stairlike structure similar to that of infectious bronchitis virus, an avian coronavirus. A proposal is presented, based on the mRNA structure, for the designation of the genes on the MHV genome. Using this proposal, the sequence differences between A59, a weakly pathogenic strain, and MHV-3, a strongly hepatotropic strain, were localized primarily in mRNA's 1 and 3, corresponding t genes A and C.     

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.     

Epstein-Barr virus RNA in Burkitt tumor tissue.

Analysis of the viral RNA in four Burkitt tumor biopsies indicates that tumor tissue contains RNA homologous to at least 3–6% of the DNA of Epstein-Barr virus (EBV). Most of these RNA species accumulate in the polyadenylated RNA fraction of Burkitt tumor tissue. Two approaches have been used to determine the location within the EBV genome of the DNA sequences which encode stable RNA in two Burkitt tumor biopsies, F and S, which contain 6–10 copies per cell of at least 80% of the EBV genome. With the first approach, ³²P-EBV DNA homologous to polyadenylated or nonpolyadenylated RNAs from the F, S or R tumors was hybridized to blots of fragments of EBV DNA. With the second approach, polyadenylated or nonpolyadenylated RNAs from the F or S tumors were hybridized to separated, labeled fragments of EBV DNA in solution. The results indicate that first, most of the viral RNA in Burkitt tumor tissue is encoded by approximately 20% of the Hsu I D fragment, 20% of the Eco RI A/Hsu I A double-cut fragment and 3% of the Hsu I B fragment of EBV DNA; second, an abundant RNA species in tumor tissue is homologous to the “additional DNA” present in the W91 and Jijoye/HR-I Burkitt tumor isolates of EBV and absent in the B95-8 virus, an isolate of EBV from outside the Burkitt endemic region; and third, there is little or no homology to other regions of the EBV genome.     

The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri).

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 X 10(-2) and 1 X 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.     

Multiple mRNA species for adenovirus type 2 polypeptides III and pVII.

Fractionation of messenger activities isolated from the cytoplasm of HeLa cells late in infection with adenovirus type 2 reveals that viral polypeptides III and pVII are each synthesized from two different-sized mRNA's. the major messenger activity for each protein has the same sedimentation rate as that previously reported by Anderson et al. (Proc. Natl. Acad. Sci. U.S.A. 71:2756-2760, 1974). The minor messenger activities for III and pVII sediment more rapidly and are not aggregates of the major mRNA's for these proteins. The two minor messenger activities cosediment with two polyadenylated RNA species which are labeled late in infection with 32P and whose molecular weights are estimated to be 2.9 x 10(6) and 2.4 x 10(6). Both of these species hybridize to adenovirus type 2 DNA specific for the mRNA family that is 3' coterminal at adenovirus type 2 map position 49.5 and the mRNA family that is 3' coterminal at 62.0. This is consistent with the possibility that these RNAs have 5'-terminal sequences identical to those of the normal mRNA's for III and pVII but are 3' coterminal at map position 62, the normal 3' terminus of the mRNA's for polypeptides II and pVI. These species are not found in polyadenylated RNA isolated from the nucleus, suggesting that the minor mRNA species are cytoplasmic RNAs.     

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.     

Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs

Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.     

Identification of a high molecular weight presumptive precursor to albumin mRNA in the nucleus of rat liver and hepatoma cell line H4AZC2.

Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.     

High molecular weight RNAs from Rous sarcoma virus and Moloney murine leukemia virus contain two subunits.

The molecular weights of the large genomic RNAs from Rous sarcoma and Moloney murine leukemia viruses were determined by a combination of sedimentation coefficients and retardation coefficients from gel electrophoresis. Six RNA standards, ranging from 0.7 X 10(6) to 5.3 X 10(6) daltons, were employed. Studies in the presence of varying concentrations of Mg2+ showed that the method provided valid molecular weights for RNAs of differing amounts of ordered structure. The molecular weight (X 10(-6)) of the high molecular weight RNA complexe from Rous sarcoma virus was 7.6 (+/-0.3) and from murine leukemia virus was 6.9 (+/-0.3). The molecular weights (X 10 (-6) of their Subunits were 3.3 (+/-0.1) and 2.8 (+/-0.2), respectively. Hence, the large complexes consisted of two, not three or more, subunits plus small associated RNAs. The high molecular weight RNA from cloned Rous sarcoma virus was heterogenous in molecular weight although the apparent molecular radius was constant; stuides were performed on subfractions of the RNA as well as on RNA from virus harvested at various time intervals. The preparations with lowest molecular weight approached a mass equal to twice that of the subunit, with hydrodynamic properties approaching those expected of normal single-stranded RNA.