RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp in vitro

Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity.     

Replication of tobacco ringspot virus. II. Differences in nucleotide sequences between the viral RNA components

Tobacco ringspot virus (TRSV)-specific double-stranded (ds)-RNA was used in competition hybridization experiments to compare the nucleotide sequences of the viral RNAs. The results indicate that the RNA of molecular weight 1.4 × 10⁶ (RNA₁) isolated from either middle or bottom component particles of TRSV have indistinguishable nucleotide sequences. However, the sequences of the RNA of molecular weight 2.3 × 10⁶ (RNA₂) are distinct from those of RNA₁ although these RNAs may have sequences of about 900 nucleotides in common.     

Isolation and characterization of a large "hairpin" segment from avian retrovirus RNA.

Avian retrovirus RNA (both 70 S and 35 S) possesses several attributes of double-stranded (ds) RNAs. Among them are an affinity for hydroxyapatite equal to that of authentic ds RNAs and exceeding that of any other single-stranded RNA tested except hnRNA, and an unexpectedly high melting temperature of some of its sequences, indicating the presence of intramolecular base-paired regions. Limited digestion with pancreatic RNase A of Prague C strain of Rous sarcoma virus as well as B77 and RAV-2 35 S RNA yielded a product that accounted for about 7% of the total viral RNA, behaved like reovirus ds RNA when chromatographed on hydroxyapatite, possessed a T_m that was similar to that of reovirus ds RNA, was almost as susceptible to RNase III as reovirus ds RNA under conditions when reovirus messenger RNA was completely resistant, and could be isolated as a relatively homogenous component following centrifugation in sucrose density gradients or electrophoresis in formamide-containing polyacrylamide gels. Its properties were consistent with the interpretation that it is a highly (but not perfectly) base-paired hairpin about 350 base pairs long. The was mapped by determining which of various size classes of poly(A)-containing fragments of viral RNA contained it and found to be located in the region between 5000 and 6000 nucleotides from the 3′-terminus of nondefective viral RNA; this region is at, or close to, the junction of the pol and env genes. The fact that the RNA of the helper virus free Bryan high-titer strain of RSV, which lacks most of the env gene, did not yield such a hairpin fragment agrees with this conclusion.     

Defective interfering particles of Sindbis virus: V. Sequence relationships between SVSTD 42 S RNA and intracellular defective viral RNAs

The annealing of Sindbis virus RNAs was studied and optimal conditions were determined (50% formamide, 6× SSC, 60°). Using these conditions, we examined the sequence relationships between various Sindbis virus ³²P-labeled single-stranded probe RNAs (42, 26, and a defective 22 S species) and five different driver double-stranded RNAs (22 S, RF-3, and the defective 18, 15, and 12 S species). It was concluded that each defective viral RNA represents a unique nonrepetitive fraction of the SV_STD genome and that all of the sequences present in the smallest defective RNA are present in the larger defective RNAs generated earlier in the passage series. Furthermore, as they decreased in size, the defective RNAs retained about 1000 nucleotides from the 26 S region of the genome, but progressively smaller amounts from the non-26 S region. These results, along with observations of other workers, are consistent with the idea that defective viral RNAs are derived from the viral genome by a progressive process of internal deletion.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

In vitro pulse-labeling of the replicative forms of bromegrass mosaic virus RNA

The in vitro pulse-labeled RNA products of a crude RNA polymerase preparation from barley leaves infected with bromegrass mosaic virus (BMV) were analyzed by electrophoresis in polyacrylamide gels.Radioactivity was incorporated in molecules having the properties of replicative form (RF), as indicated by their resistance to RNase in high salt, their dissociation by heat, their solubility in 2 M LiCl, and their electrophoretic mobility. The molecular weight of the 2 main RF components, as compared to that of reovirus-RNAs, were estimated to be 2.0 × 10⁶ and 1.5 × 10⁶ daltons, respectively, thus corresponding approximately to twice the molecular weight of the major single-stranded BMV-RNA (1.09 and 0.99 × 10⁶ and 0.75 × 10⁶).After heating at 65 ° in 89% dimethyl sulfoxide, radioactivity was transferred from the zone of double-stranded RNA to the zone of single-stranded RNA, and was associated with molecules having the migration properties of BMV-RNA components, thus showing that the label in the RF structures was incorporated into full-length molecules of viral RNA.     

Termperature-sensitive mutants of pseudorabies virus with differential effects on viral and host DNA synthesis

Mouse kidney cells infected with polyoma virus were labeled for 20 min with [5,6-³H]uridine late in infection. The rapidly-labeled RNA was extracted from whole cells and from the Hirt SDS/high salt supernatant fraction. The RNA was self-annealed and became resistant to ribonuclease digestion. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded RNA by several different criteria: resistance to RNase and to the combined effects of RNase and DNase, the characteristic buoyant density of double-stranded RNA in Cs₂SO₄, a buoyant density increase upon denaturation to that of single-stranded viral RNA marker, an inability to hybridize with complementary DNA unless denatured, and a high T_m value with a sharp transition to RNase sensitivity. Separated strands of the double-stranded RNA hybridized with high efficiency with component I of polyoma DNA. After self-annealing in formamide at low temperature, from 18 to 30% of the total rapidly-labeled viral RNA of infected cells sedimented at 11 S; 11 S corresponds in size to about 30% of the polyoma DNA. The extraction procedure for double-stranded viral RNA also yields double-stranded cellular RNA, but the double-stranded viral RNA can be further purified by using the Hirt supernatant, the RNaseresistance of the viral RNA coupled with its higher T_m, its greater sedimentation coefficient.These observations indicate that, late in infection of mouse cells, polyoma DNA is transcribed symmetrically over a considerable portion of its length, yielding self-complementary RNA that is distinguishable from the cellular self-complementary RNA.     

Comparison of oligonucleotides produced by RNase T1 digestion of 7 S RNA from avian and murine oncornaviruses and from uninfected cells

The 7 S RNA molecules isolated from Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), mouse sarcoma virus (MSV), and L cell virion (LCV) were treated with RNase T1, and the oligonucleotides produced were fractionated by two-dimensional electrophoresis. The 7 S RNAs from RSV and AMV yield identical fingerprints. Thirty-five oligonucleotides are observed in yields greater than 0.5 mole per mole RNA, and the molecules consist of about 280 nucleotides. LCV and MSV 7 S RNAs show a difference of two oligonucleotides of a total of 37. Comparison of avian and murine 7 S RNAs also shows a large number of similarities, including the 3′ terminus (U₄C₂)X_OH. The results indicate that large portions of the sequence of 7 S oncornavirus RNAs have been conserved among viruses that replicate in different hosts. Analysis of 7 S RNA from uninfected chick fibroblasts demonstrates that they contain the same molecule as that found in AMV and RSV.     

Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: selective removal of host RNA polymerase from membranes containing CCMV RNA replicase.

Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg²⁺ ions for activity. Manganese ion was a poor substitute for Mg²⁺. Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg²⁺ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.     

The ribonucleic acids of Uukuniemi virus,a noncubical tick-borne arbovirus

Sucrose gradient centrifugation of SDS-treated, purified Uukuniemi virus labeled with ${}^{32}\text{P}$, gave three reproducible peaks of single-stranded RNA, which sedimented at 27–29 S (L), 22 S (M), and 17–19 S (S). In polyacrylamide-agarose gels these RNAs separated into four peaks with apparent molecular weights of about 4.1 × 10⁶ (L), 1.9 × 10⁶ (M), 0.88 × 10⁶ (S1), and 0.78 × 10⁶ (S2), respectively. The major M and L RNAs, which represented about 75% of the total, were found in a constant molar ratio of 1:4.6.Treatment of the RNA species with 1 M formaldehyde or by heating to 100 ° did not result in a degradation of the larger to the smaller RNAs.The base compositions of the RNA species did not differ significantly from each other, but were clearly different from those of the ribosomal 28 S and 18 S RNAs extracted from host chick embryo cells.RNAs with identical sedimentation values and mobilities in gels were found from Uukuniemi virus-infected cells labeled with [${}^3\text{H}$]uridine in the presence of actinomycin D.     

Electron microscopy of viral RNA: avian tumor virus RNA.

We have investigated by electron microscopy the structure of native, partially denatured, and heated and quick-cooled RNA from avian myeloblastosis virus (AMV) and Schmidt-Ruppin Rous sarcoma virus (SR-RSV). Native 60–70 S RNA has a highly folded structure, while partially denatured 60–70 S RNA is more extended but retains much secondary and tertiary structure and has multiple free ends. 60–70 S RNA that has been heated and quick-cooled appears smaller than unheated RNA and retains some secondary structure when prepared for electron microscopy under nondenaturing conditions. The appearance of 60–70 S RNA before and after heating indicates a dissociation of the 60–70 S RNA on heating rather than a change in conformation of the RNA. Molecular weight of large subunit RNA has been determined by electron microscopy of formaldehyde-formamide treated RNA. The largest RNA subunit of AMV has a molecular weight of 2.9 × 10⁶. The largest subunit of the transforming virus SR-RSV is 20% larger with a molecular weight of 3.5 × 10⁶.     

Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19

ObjectivesThe development of a rapid diagnostic test for viable SARS-CoV-2 is important for infection control. Real-time RT-PCR assays detect non-viable virus, and cell culture differentiates viable virus but it takes several weeks and is labour-intensive. Subgenomic RNAs may reflect replication-competent virus. We therefore evaluated the usefulness of subgenomic RNAs for diagnosing viable SARS-CoV-2 in patients with COVID-19.MethodsPatients with various severities of confirmed COVID-19 were enrolled at a tertiary hospital between February and December 2020. RT-PCR assay results for genomic and subgenomic RNA of SARS-CoV-2 from nasopharyngeal swab, sputum and saliva specimens were compared with cell culture results.ResultsA total 189 specimens from 20 COVID-19 patients were tested in genomic and subgenomic PCR assays and cultured on Vero cells. Of these 189 samples, 62 (33%) gave positive culture results, 93 (49%) negative results and the remaining 34 (18%) indeterminate results. Compared with cell culture results, the sensitivities of genomic RNA and subgenomic RNA of the N and S genes were comparable at 100%, but the specificity of subgenomic RNA (N, 65% and S, 68%) was higher than that of genomic RNA (N, 23% and S, 17%, p < 0.001). The mean durations of positive culture and subgenomic RNA were 11.39 ± 10.34 and 13.75 ± 11.22 days after symptom onset (p 0.437), respectively, while that of genomic RNA was 22.85 ± 11.83 days after symptom onset (p < 0.001).DiscussionOur comparison of subgenomic RNA detection with symptom duration and SARS-CoV-2 culture positivity provides a significant advancement on the transmissibility-based approach beyond the detection of SARS-CoV-2 genomic RNA, and warrants further studies on the development of better diagnostic strategy.     

Isolation and characterization of double stranded RNA containing infectious viral genome RNA from cells infected with Semliki Forest virus

Summary A double stranded virus specific RNA sedimenting at about 19S on sucrose density gradients has been isolated from BHK-21 cells infected with Semliki Forest virus (SFV). The molecule consists of double stranded RNA (ds RNA) since it is labeled with³H-uridine, is souble in 2m LiCl, resistant against treatment with DNase and RNase at 2×SSC, hydrolyzed by alkali treatment, has a sharp thermal melting point at 89° in 1/10 SSC, and an extended appearance under non denaturing conditions in the electronmicroscope. The following findings show that it consists of intact, infectious 42S RNA similar or identical to the genome RNA of SFV complexed to a complementary 42S minus strand RNA: 1. Denaturation converts the ds RNA into molecules cosedimenting with 42S RNA isolated from SFV particles. 2. About 50 per cent of the radioactivity of³H-uridine labeled 42S RNA molecules generated from 19S ds RNA by denaturation hybridizes to 42S viral RNA. 3. The specific infectivity of denatured 19S ds RNA is about half of that of similarly treated viral 42S RNA. Further properties of this molecule are discussed.With 5 Figures     

Grouping of RNA coliphages based on analysis of the sizes of their RNAs and proteins.

In order to elucidate the intergroup relationships among four groups of RNA coliphages (RNA phages), we studied the sizes of their RNAs by measuring the sedimentation velocity of the RNA in a sucrose density gradient and the electrophoretic mobility of the RNA and those of proteins in polyacrylamide gel. The RNAs of group I, II, III, and IV phages (including serological intermediates) were found to have sedimentation coefficients of 24, 23, 27, and 28 S by sucrose density gradient centrifugation analysis and to have average molecular weights of 1.21, 1.20, 1.39, and 1.42 × 106 daltons by gel electrophoretic analysis, respectively. In the virions of group I and II phages, there were two kinds of protein (maturation protein and coat protein). In those of group III and IV phages, an additional protein, read-through (IIb or Al) protein (average molecular weights: 3.85 × 10⁴ for group III, and 3.90 × 10⁴ for group IV phages) was detected. The average molecular weights of coat protein from groups I, II, III, and IV were 1.40, 1.29, 1.69, and 1.73 × 10⁴, respectively. Those of maturation protein were 4.48, 4.45, 4.50, and 4.8 × 10⁴, respectively. Read-through protein was synthesized not only in cells infected with group III and IV phages, but also in a cell-free protein synthesizing system directed by groups III and IV phage RNAs.These results indicate that a distinct difference (about 20%) in molecular size of RNA exists between groups I and II and groups III and IV, which reflects the presence of readthrough protein in groups III and IV. The above results suggest that the molecular sizes of RNAs and virion proteins may offer a useful means for grouping RNA phages, because the present results were in good agreement with those of grouping of RNA phages based on serological property. In this respect, the serologically intermediate phages, JP34 and MX1, were classified into groups II and IV, respectively.     

A reexamination of influenza single-and double-stranded RNAs by gel electrophoresis.

Influenza virus single- and double-stranded RNAs have been examined by polyacrylamide-gel electrophoresis on slab gels. In both cases eight RNA segments have been demonstrated, and these are grouped as three large, three intermediate, and two small segments. The single-stranded RNAs were electrophoresed in gels containing 6 M urea, and the molecular weight of the entire single-stranded RNA genome of influenza virus was estimated to be 5.9 × 10⁶.     

Defective interfering particles of Semliki Forest virus generated in BHK cells do not interfere with viral RNA synthesis in Aedes albopictus cells

Defective interfering (DI) particles of Semliki Forest virus (SFV) were generated by serial high multiplicity passage of plaque-purified virus on BHK cells. Defective interfering passages of SFV depressed the synthesis of 42 and 26 S viral RNA and induced the formation of two new single-stranded RNA forms (molecular weight, 1.2 × 10⁶ and 0.56 × 10⁶) in BHK and Vero cells but not in Aedes albopictus cells. These results suggest that the invertebrate cells restricted replication of the alphavirus DI particles.     

Analysis of RNA associated with the poliovirus RNA replication complexes

Two size-classes of RNA replication complexes were isolated from the smooth microsomal fraction of poliovirus-infected HeLa cells: a complex that sediments at less than 70 S and another in the region from 100 S to 300 S. The virus-specific RNAs associated with the replication complexes were characterized by velocity sedimentation, acrylamide-agarose gel electrophoresis, and hybridization with poliovirus RNA. In vivo, the large replication complex contains predominantly single-stranded 35 S RNA, but only 8% of the RNA anneals to viral RNA. The small replication complex contains predominantly double-stranded RNA, and over 60% of this RNA anneals to viral RNA. These results suggest that the small replication complex may be the primary site of complementary RNA synthesis in the cell.About 50% of the RNA synthesized in vitro by the large replication complex is single-stranded RNA, whereas almost all of the RNA synthesized in vitro by the small replication complex is double-stranded RNA. However, between 20 and 30% of the RNA synthesized in vitro by both the large and the small replication complexes is complementary RNA. Possible reasons for the differences between the in, vivo and in vitro function of the replication complexes are discussed.