An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.     

Inhibition of arbovirus assembly by cycloheximide

Addition of cycloheximide (100 μg/ml) to cultures of chick cells infected with Semliki Forest virus (SFV) halted subsequent increase in virus titers. When added after 4 hr of infection, the drug had no effect on the rate of viral ribonucleic acid (RNA) synthesis, although marked inhibition of protein synthesis was seen. All of the previously identified forms of SFV RNA were seen in the drug-treated cells at higher concentrations than were present in untreated controls. The latter observation appeared to result from a failure to form viral “cores” or nucleocapsids in the cycloheximide-treated cells, resulting in sequestration of viral RNA intracellularly. The failure to form new virus cores was correlated with the failure of type II cytopathic vacuoles to appear in thin sections. Virus budding from the cell surface and the formation of type I cytopathic vacuoles persisted in cycloheximide-treated cells. The cellular pool of the major protein present in the virus core appeared to be small. None of this protein was found in a free pool in cytoplasm. The results indicated that, in the presence of cycloheximide, virus assembly was impaired because of the small size of the cellular pool of the major protein required for virus core formation.     

Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called “β-flap” and a C-terminal segment (designated “linker”) that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the β-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.     

Generation and suppression of microsomal ribonuclease activity after treatments with auxin and cytokinin

RNase activity was assayed in subcellular fractions of apical regions of Pisum sativum L. var. Alaska epicotyls after seedling decapitation and treatments with various growth regulators. High concentrations of applied indoleacetic acid caused a marked increase to occur in the RNase activity level associated with “heavy” microsomes, e.g., a 20-fold rise per unit RNA or protein in 3 days. This rise could be abolished by treating with the cytokinin benzyladenine along with indoleacetic acid. Nevertheless, indoleacetic acid and benzyladenine acted synergistically in their abilities to evoke swelling and net synthesis of RNA and protein. Polysomal profiles prepared after treatment with indoleacetic acid plus benzyladenine showed less degradation than profiles from any other treatment. It is concluded that auxin generates and cytokinin suppresses the activity of a particular membrane-bound RNase which can control turnover of the auxin-evoked polysomes required for growth in peas. Synergism between the two hormones in this system may be explained by the action of one to increase RNA synthesis and the other to decrease RNA destruction.     

Pheromone-encoding mRNA is transported to the yeast mating projection by specific RNP granules

Association of messenger RNAs with large complexes such as processing bodies (PBs) plays a pivotal role in regulating their translation and decay. Little is known about other possible functions of these assemblies. Exposure of haploid yeast cells, carrying mating type “a,” to “α pheromone” stimulates polarized growth resulting in a “shmoo” projection; it also induces synthesis of “a pheromone,” encoded by MFA2. In this paper, we show that, in response to α pheromone, MFA2 mRNA is assembled with two types of granules; both contain some canonical PB proteins, yet they differ in size, localization, motility, and sensitivity to cycloheximide. Remarkably, one type is involved in mRNA transport to the tip of the shmoo, whereas the other—in local translation in the shmoo. Normal assembly of these granules is critical for their movement, localization, and for mating. Thus, MFA2 mRNAs are transported to the shmoo tip, in complex with PB-like particles, where they are locally translated.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.     

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO₄ duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO₄, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization.     

ABERRANT INTRANUCLEOLAR MATURATION OF RIBOSOMAL PRECURSORS IN THE ABSENCE OF PROTEIN SYNTHESIS

To help elucidate the role of protein in the maturation of ribosomal RNA in cultured L cells, we have studied the effects of cycloheximide upon the maturation process and upon the intranucleolar ribonucleoprotein particles containing the "preribosomal RNA's." Five parameters of these particles were analyzed: (a) extractability, (b) sedimentation characteristics in sucrose gradients, (c) RNA composition, (d) buoyant density in CsCl gradients, and (e) effects of increased ionic strength on the buoyant density. When protein synthesis is inhibited, the rate of conversion of the precursor 45S ribosomal RNA is rapidly diminished, falling to less than 30% of the control rate within 1 hr. Nevertheless, in terms of the first three parameters there is no difference between control and cycloheximide nucleolar particles. However, the cycloheximide particles have a lower and more heterogeneous buoyant density and a more variable response to increased ionic strength. The results imply that the protein composition of the cycloheximide particles is different from that of particles from control cells, and that the entire protein complement is not necessary for the first cleavages in the maturation process, although it is necessary for the normal rate of processing and for the eventual appearance of both 18S and 28S rRNA in mature ribosomes.     

Protein synthesis is not required for the inhibitory effect of selenite on cell colony formation and RNA synthesis

Selenite has been shown to undergo intracellular metabolism that results in its conversion to other low molecular weight Secontaining species and also to its incorporation into a selenocysteine residue in selenoprotein. In order to investigate whether the incorporation into protein is required for the cytotoxic effects of selenite, we have examined whether inhibition of protein synthesis prevents the inhibitory effect of selenite on the ability of cells to form colonies or to synthesize RNA. We have found that treatment of HeLa cells with cycloheximide inhibited protein synthesis by >90% but had no effect on the inhibitory effect of selenite on cell colony formation or RNA synthesis. Since protein synthesis is not necessary for these cytotoxic effects of selenite they are unlikely to result from an increase in the synthesis of selenoproteins.     

Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.     

Functions of the two particles of tobacco rattle virus

Functions of long and short particles of five different tobacco rattle virus (TRV) systems were studied by complementation experiments with the corresponding long and short species of ribonucleic acid (RNA). The progeny of long RNA species alone was proteinless or “free” infectious long RNA, whereas short RNA species alone did not replicate by themselves but appeared to be dependent on long RNA for replication. When both types of RNA derived from the same isolate were inoculated together, particulate virus with long and short particles was produced in more than 50% of the resulting primary infections. These virus systems obtained by homologous complementation resembled the parent isolates in all their characteristics. In addition, heterologous complementation tests were performed with long and short RNA, each derived from another isolate. Heterologous interaction could be observed in only 2 out of 20 possible combinations. As a result, two “mixed” TRV systems with respect to their particle length distributions were obtained, since their long and short particles resembled the ones from the other isolate. The symptoms produced by these mixed viruses were determined by the corresponding long RNA and appeared not to be influenced by the heterologous short one. However, the protein coat of both particles of the “mixed” viruses was specified by the corresponding noninfectious short RNA. Therefore, TRV is a system of at least two functionally defective and mutually complementing components which appear to be specialized in early and late functions.     

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.     

Effects of juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

Changes in cellulase and pectinase activities in fruit tissues and separation zones of citrus treated with cycloheximide

Cellulase activity increased in separation-zone tissues 1 day after “Valencia” orange (Citrus sinensis [L.] Osbeck) was treated with 20 micrograms per milliliter cycloheximide. Exocellulase was detected only in the separation zones of treated fruit, whereas endocellulase was present in zones from both treated and control fruit. Endocellulase activity in separation-zone tissue of treated fruit was nearly three times as great as that in control tissues. Cellulase activity was restricted to separation-zone tissue. Pectinase and an albedo-macerating factor activity were very low and were not influenced by the treatment. The cycloheximide effect in these experiments was apparently caused by ethylene produced by wound tissue.     

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O₂ concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered.     

Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.