Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

Analysis of Arbovirus Ribonucleic Acid Forms by Polyacrylamide Gel Electrophoresis

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Reovirus Replicase-Directed Synthesis of Double-Stranded Ribonucleic Acid

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand (³H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of ³H-cytidine triphosphate (or ³H-uridine triphosphate) and adenine triphosphate-α-³²P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3′-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of ³H to ³²P in these fragments indicated that RNA synthesis proceeded from the 5′ to 3′ terminus.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV^Heavy, is more infectious on a per-particle basis than SINV^Light. SINV produced in mosquito cell lines (SINV^C6/36) exhibited particle-to-PFU ratios similar to those observed for SINV^Heavy. In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV^Heavy or SINV^C6/36 than following infection with SINV^Light, due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV^Heavy and SINV^C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles.     

Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver

The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm² with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA.     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

Characterization of the RNA Required for Biosynthesis of delta-Aminolevulinic Acid from Glutamate : Purification by Anticodon-Based Affinity Chromatography and Determination That the UUC Glutamate Anticodon Is a General Requirement for Function in ALA Biosynthesis

The heme and chlorophyll precursor δ-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3′ terminus of yeast tRNA^Phe(GAA) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA^Glu(UUC) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A₂₆₀ unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae.     

Differential synthesis of RNA in self- and cross-pollinated styles of Petunia hybrida L.

Summary Styles of Petunia hybrida were pollinated (selfed or crossed) and pollentubes were allowed to grow in the presence of ³H-orotic acid. After 24 h RNA was extracted using the cold phenol method and separated on a tandem polyacrylamide gel. Results show that: 1. the synthesis of RNA is more enhanced after cross- than after selfpollination and 2. a restricted number of RNA peaks is responsible for this difference; this RNA has a molecular weight different from r-RNA and t-RNA and could be messenger-like of nature. Results indicate a differential gene expression correlated with rejection or acceptance of the pollentubes by the style.     

Characterization of segmented double-helical RNA from bacteriophage phi6

The nucleic acid component of bacteriophage φ6 is characterized as a double stranded RNA molecule with a buoyant density of 1.605 g/cm³ and nucleotide composition of C, 27.3%; A, 21.8%; G, 28.9%; and U, 22.0%. The hyperchromicity profile in 0.1 × SSC (SSC is 0.15 m-NaCl, 0.015 m-sodium citrate) demonstrated a rapid increase with a T_m value of 91 °C. The nucleic acid was resistant to degradation by DNase, spleen phosphodiesterase and pancreatic RNase in 2 × SSC buffer but sensitive to degradation by venom phosphodiesterase, pancreatic RNase in 0.01 × SSC and hydrolysis in KOH. Three distinct double stranded RNA species of 2.2, 2.8 and 4.5 × 10⁶ daltons were observed after rate zonal centrifugation, polyacrylamide gel electrophoresis and electron microscopy. This communication therefore presents data establishing a new class of double stranded RNA bacteriophage.     

RNA sequence determination in the nanogram range by a combination of in vitro labeling procedures.

Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′-³²P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T₁ and RNase A, respectively. These digests were again ³²P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-chloroform extraction step during 5′-end labeling with polynucleotide kinase and [γ-³²P]ATP; (b) ZnSO₄ inactivation of RNase T₁ results in a highly efficient procedure for 3′-end labeling with T4 ligase and [5′-³²P]pCp; and (c) a rapid 4-min procedure for variable quantity range of ¹²⁵I and RNA results in a qualitative and quantitative sample for high-molecular weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

Analysis of purified tRNA species by polyacrylamide gel electrophoresis

Six purified amino acid acceptor tRNA species were examined by polyacrylamide gel electrophoresis. Small differences in migration were observed under conditions that preserve the conformation of tRNA. When tRNA was heated in the presence of either 10 mM acetate or EDTA at 60° a change in migration was observed for tRNA^Glu. No difference in migration was seen between Val-tRNA^Val and tRNA^Val. When tRNA was denatured by heating in 4M urea and applied to a gel containing the same amount of urea, all tRNA species migrated approximately the same distance with the exception of tRNA^{Leu V}, which showed an appreciable slower migration. From the difference in migration of tRNA^{Leu V} as compared to tRNA^Val and 5 S RNA, the difference in chain length between tRNA^Val and tRNA^{Leu V} was estimated to be approximately 9 nucleotides.     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.