Properties of Single- and Double-stranded Ribonucleic Acid from Barley Plants Infected with Bromegrass Mosaic Virus

Incorporation of ³²P into nucleic acids in barley plants infected with bromegrass mosaic virus (BMV) was analyzed by chromatography on methylated albumin kieselguhr (MAK) columns. Treatment with actinomycin D reduced the synthesis of ribosomal ribonucleic acid (RNA) to low levels and allowed the detection of the three components of BMV-RNA in vivo. The kinetic study on ³²P incorporation into these BMV-RNA components suggested that a single cleavage occurred in some of the intact RNA shortly after completion of its synthesis, giving rise to the small and medium components. Chromatographic analyses also revealed a double-stranded, ribonuclease-resistant RNA which has been purified by differential extraction, sucrose-density gradient centrifugation, and MAK column chromatography. This RNA sediments at approximately 14S, is alkali-labile, and has a sharp thermal transition with a T_m of 96 C in 0.1 × standard saline citrate buffer, as determined by susceptibility to ribonuclease. The RNA is absent in uninfected barley plants.     

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA^{Tyr I}. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.     

Affinity labeling of specific regions of 23 S RNA by reaction of N-bromoacetyl-phenylalanyl-transfer RNA with Escherichia coli ribosomes

Reaction of the affinity-labeling reagent N-bromoacetyl-[¹⁴C]phenylalanyl-tRNA with Escherichia coli ribosomes results in covalent labeling of 23 S ribosomal RNA in addition to the previously reported labeling of ribosomal proteins. The reaction with the 23 S RNA is absolutely dependent on the presence of messenger RNA. Covalent attachment of the affinity label to 23 S RNA was demonstrated by its integrity in strongly dissociating solvents, and the conversion of the labeled material to small oligonucleotides by ribonuclease treatment. After digestion of labeled 23 S RNA with T₁ ribonuclease, the radioactivity is found mainly in two oligonucleotide fragments. These results support models in which both ribosomal RNA and ribosomal protein contribute to the structure of the region of the ribosome surrounding the peptidyl transferase center.     

Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA

Viral resistance can be effectively induced in transgenic plants through their silencing machinery. Thus, we designed nine short hairpin RNAs (shRNA) constructs to target nuclear inclusion protein b (NIb), helper component proteinase (HC-Pro), cylindrical inclusion protein (CI) and viral protein genome linked (VPg) genes of Potato virus Y (PVY^N) and Tobacco etch virus (TEV-SD1). The shRNAs were completely complementary to the genes of PVY^N, and contained 1–3 nt mismatches to the genes of TEV-SD1. To study the specificity of gene silencing in shRNA-mediated viral resistance, the constructs were introduced into tobacco plants. The results of viral resistance assay revealed that these nine kinds of transgenic tobacco plants can effectively induce viral resistance against both PVY^N and TEV-SD1, and the shRNA construct targeting the NIb gene showed higher silencing efficiency. Northern blot and short interfering RNA (siRNA) analyses demonstrated that the viral resistance can be attributed to the degradation of the target RNA through the RNA silencing system. Correlation analysis of siRNA sequence characteristics with its activity suggested that the secondary structure stability of the antisense strand did not influence siRNA activity; 1 to 3 nt 5’ end of the sense strand caused a significant effect on siRNA activity where the first base such as U was favourable for silencing; the base mismatch between the siRNA and the target gene may be more tolerated in the 5’ end.     

Characterization of the ribonuclease activity on the skin surface

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin^® although not by the ribonuclease inhibitor SUPERase· In?. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.     

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral E^rns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic.     

Studies on the physicochemical properties of the new preparation deltaferon as a component of a molecular construct

A molecular construct of a spherical shape containing double-stranded RNA (dsRNA) in the center, coated by a spermidine-polyglucine coat, and carrying recombinant deltaferon—an analogue of recombinant interferon-gamma—has been designed. It is shown that deltaferon in this construct retains its specific activity at a level of 9 × 10⁴ U/mg. It was found that deltaferon and dsRNA in the construct were characterized by enhanced resistance to proteases (trypsin) and nucleases (E. coli ribonuclease) as compared to free deltaferon and dsRNA. An increased resistance of deltaferon in the construct to environmental temperature during storage was also demonstrated.     

Replication of Western Equine Encephalomyelitis Virus II. Cytoplasmic Structure Involved in the Synthesis and Development of the Virions

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with ³H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to ³H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of ¹⁴C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with ³H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.     

Nucleotide Composition of RNA hybridized to Homologous DNA from Cells transformed by Avian Tumour Viruses

PART of the evidence which indicates that RNA tumour viruses replicate through a DNA intermediate¹ was the detection of DNA which is complementary to the viral RNA in leukaemic cells transformed by avian myeloblastosis virus (AMV)² and in cells transformed in vitro by avian sarcoma viruses, Schmidt-Ruppin (SR-RSV) and B-77 (ref. 3). If this DNA serves as a template for the viral RNA, it must be a copy of the entire viral genome. One of the necessary requirements for this function is that the homologous DNA has the same nucleotide composition as the viral RNA. In this study, the average base composition of the RNA which had been hybridized to homologous DNA from transformed cells was compared with the base composition of the input viral RNA. Two experimental conditions had to be met: (1) the recovery of all the ribonucleotides which had been hybridized and (2) the absence of partially hybridized ribonucleotide sequences. The first requirement called for the deletion of the treatment of DNA-RNA hybrids with pancreatic ribonuclease fraction A and ribonuclease T₁ which had been used in our previous experiments because such a treatment can cause the non-random loss of hybridized nucleotides⁴. The second requirement called for a hybridization and washing procedure in which only specifically hybridized ribonucleotide sequences would remain bound to the filters. Both of these conditions were met by using fragmented viral RNA and a modified washing procedure which excluded the use of ribonuclease. The results show that the average nucleotide composition of the hybridized RNA is identical to that of the input viral RNA.     

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-³H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3′-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.     

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSs^TYRV was just as strong as those by NSs^TSWV and NSs^GRSV, even though NSs^TYRV was expressed in lower amounts. Using the system established, a set of selected NSs^TSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSs^TSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.     

DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Some properties of influenza virus nucleocapsids

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.     

Identification of a conserved RNA replication element (cre) within the 3Dpol-coding sequence of hepatoviruses

Internally located, cis-acting RNA replication elements (cre) have been identified within the genomes of viruses representing each of the major picornavirus genera (Enterovirus, Rhinovirus, Aphthovirus, and Cardiovirus) except Hepatovirus. Previous efforts to identify a stem-loop structure with cre function in hepatitis A virus (HAV), the type species of this genus, by phylogenetic analyses or thermodynamic predictions have not succeeded. However, a region of markedly suppressed synonymous codon variability was identified in alignments of HAV sequences near the 5′ end of the 3D^pol-coding sequence of HAV, consistent with noncoding constraints imposed by an underlying RNA secondary structure. Subsequent MFOLD predictions identified a 110-nucleotide (nt) complex stem-loop in this region with a typical AAACA/G cre motif in its top loop. A potentially homologous RNA structure was identified in this region of the avian encephalitis virus genome, despite little nucleotide sequence relatedness between it and HAV. Mutations that disrupted secondary RNA structure or the AAACA/G motif, without altering the amino acid sequence of 3D^pol, ablated replication of a subgenomic HAV replicon in transfected human hepatoma cells. Replication competence could be rescued by reinsertion of the native 110-nt stem-loop structure (but not an abbreviated 45-nt stem-loop) upstream of the HAV coding sequence in the replicon. These results suggest that this stem-loop is functionally similar to cre elements of other picornaviruses and likely involved in templating VPg uridylylation as in other picornaviruses, despite its significantly larger size and lower free folding energy.     

The genome of Uukuniemi virus consists of three unique RNA segments

The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of ³²P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each RNA species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5′ terminal pppAp … structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5′ terminal “cap” or 3′ terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.     

Biochemical characterization of the RNA-hydrolytic activity of a pumpkin 2S albumin

A pumpkin 2S albumin with ribonuclease (RNase) activity was purified from pumpkin seeds (Cucurbita sp.) by liquid chromatographic techniques. It manifested potent RNase activity toward baker’s yeast RNA and calf liver RNA, and some polyhomoribonucleotides, including poly(A), poly(U) and poly(C) but not poly(G). Moreover, it was able to hydrolyze total RNA of both animal and plant origins. Ions such as Na⁺, Mg²⁺, Ca²⁺, and Zn²⁺ inhibited its RNase activity. Since RNase activity has not been previously reported in 2S albumins, this work may shed further light on the biological importance of this group of proteins.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of <Emphasis Type="Italic">Celosia cristata</Emphasis>

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled p BlueScript SK⁺ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower prote in concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.