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As an expert in the field of drug design and discovery, I tried, in this up-to-date perspective or commentary article, to recap and shed light on the previous and latest revolutionary strategies employed in medicinal and therapeutic chemistry to target the principal viral weapon used by virulent RNA viruses (e.g., the severe acute respiratory syndrome coronavirus 2 "SARS-CoV-2") to infect humans and spread infections, the genomic RNA strands. These strategies act by taking advantage of the weakness points of this attractive bioweapon to disable or attack it (itself), accordingly stop the entire viral reproduction, and effectively end the severe microbial infections such as the coronavirus disease 2019 (COVID-19). The generation of respective slightly falsely-weaved RNA strands, either endogenously or exogenously, is the principal key for designing most of these therapeutic approaches., (© 2023 John Wiley & Sons Ltd.)

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion., (© 2021 Zheng et al.)

Nearly 200 distinct chemical modifications of RNAs have been discovered to date. Their analysis via direct methods has been possible in abundant RNA species, such as ribosomal, transfer or viral RNA, since several decades. However, their analysis in less abundant RNAs species, especially cellular messenger RNAs, was rendered possible only recently with the advent of high throughput sequencing techniques. Given the growing biomedical interest of the proteins that write, erase and read RNA modifications, ingenious new methods to enrich and identify RNA modifications at base resolution have been implemented, and more efforts are underway to render them more quantitative. Here, we review several crucial modification-specific (bio)chemical approaches and discuss their advantages and shortcomings for exploring the epitranscriptome., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.

Competing Interests: The authors declare no conflict of interest.

The folding landscape for an RNA sequence contains many diverse structures and motifs, which are often sampled rather than completely explored. Today's supercomputers make the complete enumeration of all possible folds for an RNA and a detailed description of the RNA folding landscape a more feasible task. This chapter provides protocols for using the Crumple folding algorithm, an efficient tool to generate all possible non-pseudoknotted folds for an RNA sequence. Crumple in conjunction with Sliding Windows and Assembly can incorporate experimental constraints on the global features of an RNA, such as the minimum number and lengths of helices, which may be determined by crystallography or cryo-electron microscopy. This complete enumeration method is independent of free-energy minimization and allows the user to incorporate experimental data such as chemical probing, SELEX data on RNA-protein binding motifs, and phylogenetic covariation.

The innate immune system uses pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) to recognize conserved pathogen-associated molecular patterns (PAMPs) expressed by microbes and to activate the initial phase of immune response. Both bacterial and viral nucleic acids activate TLRs resulting in cytokines and interferon production. Recent studies indicated that unmodified small interfering RNAs (siRNAs) corresponding to either mammalian (self) or microbial (non-self) RNA sequences can activate immunity through TLR7/8 in a sequence-dependent manner. Hence, the use siRNAs in humans will require understanding the mechanisms involved in the discrimination between self and non-self RNAs. In the case where immunostimulation is not wanted, chemical modifications can prevent immune activation and reduce off-target effects, while preserving siRNA silencing potency. Interestingly, 2'-O-methyl modified RNAs not only evade immune sensing, but antagonize with immunostimulatory single- or double-stranded siRNAs to activate innate immunity. This review highlights the recent progress in understanding the siRNA sensing by innate immunity and presents a range of strategies allowing either the design of siRNAs with minimal or maximal immunostimulatory potency for therapeutic applications.

RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated. In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity. In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process.

Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3' one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3'-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (-)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (-)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.

Internally located, cis-acting RNA replication elements, termed cres, are essential for replication of the genomes of picornaviruses such as human rhinovirus 14 (HRV-14) and poliovirus because they template uridylylation of the protein primer, VPg, by the polymerase 3D(pol). These cres form stem-loop structures sharing a common loop motif, and the HRV-14 cre can substitute functionally for the poliovirus cre in both uridylylation in vitro and RNA replication in vivo. We show, however, that the poliovirus cre is unable to support HRV-14 RNA replication. This lack of complementation maps to the stem of the poliovirus cre and was reversed by single nucleotide substitutions in the stem as well as the base of the loop. Replication-competent, revertant viruses rescued from dicistronic HRV-14 RNAs containing the poliovirus cre, or a chimeric cre containing the poliovirus stem, contained adaptive amino acid substitutions. These mapped to the surface of both the polymerase 3D(pol), at the tip of the "thumb" domain, and the protease 3C(pro), on the side opposing the active site and near the end of an extended strand segment implicated previously in RNA binding. These mutations substantially enhanced replication competence when introduced into HRV-14 RNAs containing the poliovirus cre, and they were additive in their effects. The data support a model in which 3CD or its derivatives 3C(pro) and 3D(pol) interact directly with the stem of the cre during uridylylation of VPg.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) initiates RNA synthesis in vivo by a de novo mechanism. In vitro, however, the HCV RdRp can initiate de novo or extend from a primed template. A novel beta-loop near the RdRp active site was previously found to prevent the use of primed templates. We found that, in addition to the beta-loop, the C-terminal tail of the HCV RdRp and the de novo initiation GTP are required to exclude the use of primed-templates. GTP binding to the NTPi site of the HCV RdRp orchestrates the participation of other structures. The interactions of the beta-loop, C-terminal tail, and GTP provide an elegant solution to ensure de novo initiation of HCV RNA synthesis.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.

A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe. tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe.

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA(Pro)-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA(Pro) was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA(Pro) backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.

Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.

We report the sequence, structural features, and self-cleaving activity of the small circular RNA (sc-RNA) associated with rice yellow mottle sobemovirus (RYMV). At 220 nucleotides, the RYMV sc-RNA represents the smallest naturally occurring viroid-like RNA currently documented in the literature. It is similar to other circular satellite RNAs (sat-RNAs) and viroids in being G-C-rich with a high level of self-complementarity. The predicted native structure is essentially a rod with one branched terminus. A region of the RYMV sc-RNA, constituting 24% of the sequence, exhibits 89% identity to the sat-RNA associated with the Australasian isolates of lucerne transient streak sobemovirus. This region is also structurally similar in all three RNAs in that it forms the left terminus of each rod. Dimeric runoff transcripts of cloned RYMV sc-RNA undergo efficient autocatalytic in vitro cleavage in the (+) but not the (-) polarity. Analysis of the (+) sequence indicates the presence of a hammerhead ribozyme resembling that of carnation small retroviroid-like RNA and the genomic satellite transcript of newt. Inefficient cleavage of (+) monomeric transcripts, and a short stem III in the hammerhead, are features consistent with a double-hammerhead mode of self-cleavage. The presence of sat-RNA and retroviroid-like structures within a single RNA suggests a possible role for the RYMV sc-RNA as an evolutionary intermediate between these subviral RNAs.

Initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) occurs by extension from the 3' end of a cellular tRNA complexed to the primer binding site (PBS) located near the 5' end of the viral RNA genome. Although the PBSs for all naturally occurring HIV-1 viruses are complementary to the 3'-terminal 18 nucleotides of tRNA(Lys)3, we identified an HIV-1 virus which contained a PBS complementary to the 3' nucleotides of tRNA(Met); the PBS of this virus was not stable upon extended culture and reverted back to the wild type (S.-M. Kang, J. K. Wakefield, and C. D. Morrow, Virology 222:401-414, 1996). To further characterize the virus with a PBS complementary to tRNA(Met), a DNA fragment encompassing the PBS and U5 region from this proviral genome was substituted for the same region in the infectious HIV-1 proviral clone [named pHXB2(AC-Met)]. Three additional proviral genomes were also created: pHXB2(Met), which is isogenic with pHXB2 except for the PBS complementary to tRNA(Met); pHXB2(Met-AC-Met), which contains the PBS sequence complementary to the 3'-terminal nucleotides and the sequence upstream of this PBS in U5 complementary to the anticodon region of tRNA(Met); and pHXB2(Met-C-Met), which contains two G-to-C changes predicted to disrupt complementarity within the tRNA(Met) anticodon region. Viruses derived from the transfection of these proviral genomes were infectious, although the appearance of the viruses was delayed compared to that of the wild-type virus. PCR amplification and DNA sequence analysis of the PBS regions from proviral genomes revealed that the PBSs from viruses derived from pHXB2(Met) and pHXB2(AC-Met) reverted back to the wild type by days 16 and 44 postcoculture, respectively. Two new, novel mutant viruses were identified among viruses derived from pHXB2(Met-C-Met) at day 35 postcoculture: one contained a PBS complementary to tRNA(Lys)1,2, while the second maintained a PBS complementary to tRNA(Met) but contained a 26-nucleotide deletion in U5 upstream of the anticodon-complementary region. By day 125 postcoculture, the PBS in the virus from this culture had reverted back to the wild type, complementary to tRNA(Lys)3. In contrast, the viruses derived from pHXB2(Met-AC-Met) stably maintained a PBS complementary to tRNA(Met) during the 125-day culture period examined. The results of these studies support the idea that HIV-1 can maintain a PBS complementary to alternative tRNAs provided that the appropriate complementarity exists between the U5-PBS region of the viral RNA genome and the tRNA molecule used to initiate reverse transcription.

The mechanism of inhibition of HIVBa-L replication by interleukin 10 (IL-10) in primary monocytes and macrophages at various stages of maturation was investigated using semiquantitative PCR for reverse-transcribed HIV DNA, and Northern hybridization for HIV mRNA expression in comparison with extracellular p24 antigen. Pretreatment of monocytes with IL-10 markedly inhibited expression of both unspliced and spliced HIV RNA, reaching a nadir at 7 days and recovering to normal levels by 10 days after a single application. The optimum inhibitory concentration was 25 ng/ml. Less inhibition of HIV RNA expression was observed when IL-10 was added after HIV infection of monocytes and the inhibitory effect progressively declined to negligible levels as monocytes matured into macrophages over 10 days. IL-10 also downregulated the expression of cellular genes, including the transferrin receptor, 28S rRNA, and GAPDH. The kinetics of the inhibition of cellular mRNAs correlated with the inhibition of HIV RNA and also declined as monocytes matured into macrophages. In contrast, IL-10 did not inhibit cellular mRNA expression in the macrophage cell line THP-1. Neutralizing polyclonal antibody to IL-10 reversed all its inhibitory effects. Interaction of IL-10 and TNF-alpha in combination were generally antagonistic in their effects on HIV transcription. IL-10 prevented stimulation of HIV RNA expression by TNF-alpha after preincubation with monocytes for 48 hr. IL-10 had no effect on the levels of HIV cDNA or the process of initiation and completion of reverse transcription. The inhibitory effect of IL-10 on HIV replication in maturing monocytes was probably mediated mainly by inhibition of cellular gene expression and inhibition of maturation of monocytes into macrophages and their activation, with consequent downregulation of HIV mRNA.

The NS1 protein of influenza A virus has the unique property of binding to three apparently different RNAs: poly A; a stem-bulge in U6 small nuclear RNA; and double-stranded RNA. One of our major goals is to determine how the NS1 protein recognizes and binds to its several RNA targets. As the first step for conducting structural studies, we have succeeded in identifying a fragment of the NS1 protein that possesses all the RNA-binding activities of the full-length protein. The RNA-binding fragment consists of the 73 amino-terminal amino acids of the protein. We have developed procedures for obtaining large amounts of the polypeptide in pure form. This has enabled us to establish the RNA-binding properties of this polypeptide and to demonstrate that it retains the ability to dimerize exhibited by the full-length protein. In addition, far-UV CD spectroscopy indicates that this RNA-binding polypeptide is largely (approximately 80%) helical, suggesting that the mode of dimerization of the NS1 protein and of its interaction with RNA is mediated, at least in part, by helices.

A synthetic 67-nt RNA substrate, containing a 32P-labeled cap-1 structure (m7G32pppGm) was specifically cleaved by the influenza virus RNA polymerase (EC 2.7.7.48) to yield a single capped 11-nt fragment capable of directly priming transcription. An analysis of systematic truncations of this RNA substrate demonstrated that an additional nucleotide beyond this cleavage site was required for cleavage. The minimal RNA chain length required for priming activity was found to be 9 nt, while in contrast an RNA chain length of at least 4 nt was required for efficient binding to the viral polymerase. On the basis of these chain length requirements we show that a pool of capped oligonucleotides too short to prime transcription, but long enough to bind with high affinity to the viral polymerase, are potent inhibitors of cap-dependent transcription in vitro.

Turnip crinkle virus (TCV) is associated with many subviral RNAs including satellite (sat-) RNAs which require a helper virus for infectivity. When plants were inoculated with TCV and transcripts of TCV sat-RNA C containing deletions of 3 to 8 nucleotides beginning at position 100 and extending toward the 5' end, some of the sat-RNA isolated from plants migrated more slowly than expected on denaturing polyacrylamide gels. Cleavage of the sat-RNA into two segments by digestion with RNase H following hybridization to an oligonucleotide complementary to internal sat-RNA sequence indicated that the 5' one-third of the molecule was involved in the abnormal gel migration. Sat-RNAs derived from transcripts with a deletion of bases in position 96-100 were cloned. Sequencing of the cDNAs revealed that the aberrant migration of the sat-RNAs was due to the presence of variable lengths of poly(U) 10 nucleotides downstream from the deletion at a position which already contained five U residues. Deletions extending toward the 3' end in the same region did not result in poly(U) additions. Mutations in the original five U residues along with the 5' deletions also did not lead to poly(U) additions. The insertion of poly(U) in TCV sat-RNA C may be a new example of replicase stuttering with the distinction that it only occurs following specific upstream mutations.

A computer program is presented which determines the secondary structure of linear RNA molecules by simulating a hypothetical process of folding. This process implies the concept of 'nucleation centres', regions in RNA which locally trigger the folding. During the simulation, the RNA is allowed to fold into pseudoknotted structures, unlike all other programs predicting RNA secondary structure. The simulation uses published, experimentally determined free energy values for nearest neighbour base pair stackings and loop regions, except for new extrapolated values for loops larger than seven nucleotides. The free energy value for a loop arising from pseudoknot formation is set to a single, estimated value of 4.2 kcal/mole. Especially in the case of long RNA sequences, our program appears superior to other secondary structure predicting programs described so far, as tests on tRNAs, the LSU intron of Tetrahymena thermophila and a number of plant viral RNAs show. In addition, pseudoknotted structures are often predicted successfully. The program is written in mainframe APL and is adapted to run on IBM compatible PCs, Atari ST and Macintosh personal computers. On an 8 MHz 8088 standard PC without coprocessor, using STSC APL, it folds a sequence of 700 nucleotides in one and a half hour.

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.

Bound iodine is released from radioiodinated nucleotides in polymers exposed to sodium bisulfite. The rate of bisulfite-catalyzed deiodination of pyrimidines can be controlled both by change of temperature of pH and is also dependent on the molecular association of the nucleotide. The rate of release of iodine from iodocytidine in polycytidylate is greater than the rate of elimination from RNA. Experiments testing the influence of base-pairing of the iodopyrimidines in synthetic polynucleotides showed that pairing of the substituted nucleotide protected the iodine bond. The rates of bisulfite-catalyzed deiodination of several radioiodinated RNAs were measured. The action of bisulfite on all single stranded RNAs tested was multiphasic consisting of a rapid early deiodination reaction supplanted by a slower phase which was followed by reacceleration of release. The release of iodine from double stranded RNA and DNA-RNA duplexes was retarded in comparison with the release from ribosomal and messenger RNA fractions. The deiodination profiles of single and double stranded RNA suggested that the intermediate stage iodine release is governed by melting of paired zones of low stability. Late release may result from destablization of the molecule through the addition of bisulfite to the pyrimidine ring or deamination. The effect of several substances expected to complex with polynucleotides was tested. Acridine orange and ethidium bromide increased loss of iodine from ribosomal RNA but slightly decreased elimination from double stranded viral RNA. A basic protein fraction isolated from ribosomal particles accelerated the deiodination of ribosomal RNA. While the destabilization caused by this protein fraction was greater than that caused by an equal amount of albumin, as tested the effect was non-specific. The results show that a change in sensitivity to chemical deiodination may folow the interaction of small amounts of protein with polynucleotides.

The conditions for acidic denaturation of double stranded RNA were found. Under these conditions a limited degradation of high molecular weight viral RNA took place. This degradation was determined by the degree of fragmentation and loss of infectivity at acidic conditions. It was found that acidic denaturation of RNA in the solutions of low ionic strength was accompanied by a considerable increase of sedimentation coefficient. Under these conditions the coefficients of sedimentation and molecular weights of RNAs studied are connected by the following function S20=2.84-10(-2) Mr0.689. The conclusion has been drawn that the sedimentation under the conditions for acidic denaturation could be used both for molecular weight determination and the practical preparation of unaggregated strands of RNA.

The buoyant densities in Cs2SO4 of the double-stranded RNA from the bacteriophage phi 6 and the virus-like particles of Helminthosporium maydis and Penicillium chrysogenum were determined by a Taylor series expansion and by the position and slope of the gradient relative to the isopycnic position (hinge point method). Buoyant densities for the three types of double-stranded RNA calculated by the two methods were, respectively, 1.6089 and 1.6083, 1.6065 and 1.6059 and 1.6057 and 1.6050.

The genome structure of different A(H1N1) and A(H3N2) influenza virus strains isolated from 1977 to 1980 was analysed by electrophoresis of single-stranded RNA and of the double-stranded RNA obtained by hybridization with complementary RNA of the A/Stavropol 188/79 (H3N2) strain. The differences in genome structure are insignificant within the A(H1N1) subtype, and considerable (involving almost all the 8 RNA fragments) between the two A(H3N2) strains (A/Texas 1/77 and A/Stavropol 188/79). The great differences between the genomes of A(H1N1) and A(H3N2) strains do not exclude certain genetic relationships between these viruses.

Double-stranded RNA, made as an intermediary substance in the replication of most, if not all, viruses, may play a much more important role in the pathogenesis and the recovery from virus infections than has hitherto been suspected. Apparently, dsRNA is used by both the challenge virus and the host cell in an attempt to gain "molecular control." Double-stranded RNA exerts a set of effects, which may be well balanced, not only at the level of the individual cell but also at the complex assemblage of these cells termed the organism (Fig. 1). In the cell, interferon synthesis is triggered, although interferon mRNA translation may not occur if dsRNA shuts off protein synthesis too quickly. In the whole organism, the disease severity will depend on how certain toxic reactions evoked by infection (such as cell necrosis and fever) are counterbalanced by an increase in the host defense mechanisms (for example, immune responsiveness and interferon production). Many aspects of the response, relating to either progress of, or recovery from, the disease, can be explained on the basis of a dsRNA. In addition to drawing attention to the biodynamic role of dsRNA, our hypothesis suggests specific experimental vectors designed to enhance our information on the molecular basis of the morbid process which occurs with viral infection. Finally, we suggest that, although the dsRNA molecule may be viewed as a rather simple unit structure, the opportunity for further diversity in the biological activity of a given dsRNA molecule always exists. Namely, each deviation from a perfectly double-helical arrangement introduces the possibility for emphasizing one biological reactivity at the expense of another. This latter structure-activity property may partially account for the extreme apparent diversity, commonly encountered, in the presentations of virologic illness. Appendix note added in proof. Subsequent to submission of this text, we have found that the potent mitogen effect of dsRNA for lymphocytes (murine and human) is also exquisitively sensitive to the fidelity in base pairing of the input polymer pair (59). For example, infrequent "loops" (one nucleotide per 20 base pairs) in an otherwise perfectly helical rI(n) (.) rC(n) molecule [for example, rI(n) (.) r(C(19,)U)(n)] strongly changes its mitogenic properties. This observation, which supports our thesis that a "fine structure" term can be developed for other reactions triggered by dsRNA's in biological systems, emphasizes that diverse biological effects may be encountered with an ostensibly uniform family of dsRNA's.

Fast algorithms for analysing sequence data are presented. An algorithm for strict homologies finds all common subsequences of length greater than or equal to 6 in two given sequences. With it, nucleic acid pieces five thousand nucleotides long can be compared in five seconds on CDC 6600. Secondary structure algorithms generate the N most stable secondary structures of an RNA molecule, taking into account all loop contributions, and the formation of all possible base-pairs in stems, including odd pairs (G.G., C.U., etc.). They allow a typical 100-nucleotide sequence to be analysed in 10 seconds. The homology and secondary structure programs are respectively illustrated with a comparison of two phage genomes, and a discussion of Drosophila melanogaster 55 RNA folding.

A mode of action of RNase CI toward the various native RNAs was examined. The mixture of tRNA from Thermus thermophilus HB8 were exhaustively cleaved by RNase CI to produce 90% of acid-soluble fraction. Among the minor nucleotides s2T were recognized by this enzyme. RNase CI generates a linear viroid molecule by introducing one single nick into viroid circular molecule. Double stranded RNA from RNA virus, such as Cytoplasmic polyhedrosis virus (CPV) from silk worm, was cleaved at some specific sites to produce more than ten of the discrete bands electrophoretically.

Fragmented 125I-labelled RNA from RNA tumor viruses was hybridized to unlabelled RNA from cells, viruses, and homoribopolymers. The viral RNA interacted with all RNA tested, except for certain homoribopolymers. Complex formation with unlabelled RNA was verified by nuclease resistance, buoyant density measurements, and thermal stability in solutions of different ionic strength. The RNAase-resistant complex involved 20-30% of the sequences in the 125I-labelled viral RNA and formed preferentially with nuclear RNA of cells. 125I-labelled hemoglobin mRNA, 125I-labelled immunoglobulin light chain (lambda2) mRNA, or 125I-labelled viral RNA from encephalomyocarditis virus (EMC) dit not from RNAase-resistant complexes with unlabelled cellular RNA.

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.

We describe a program for the analysis of RNA secondary structure. There are two new features in this program. (i) To get vector speeds on a vector pipeline machine (such as Cray X-MP/24) we have vectorized the secondary structure dynamic algorithm. (ii) The statistical significance of a locally 'optimal' secondary structure is assessed by a Monte Carlo method. The results can be depicted graphically including profiles of the stability of local secondary structures and the distribution of the potentially significant secondary structures in the RNA molecules. Interesting regions where both the potentially significant secondary structures and 'open' structures (single-stranded coils) occur can be identified by the plots mentioned above. Furthermore, the speed of the vectorized code allows repeated Monte Carlo simulations with different overlapping window sizes. Thus, the optimal size of the significant secondary structure occurring in the interesting region can be assessed by repeating the Monte Carlo simulation. The power of the program is demonstrated in the analysis of local secondary structures of human T-cell lymphotrophic virus type III (HIV).

Annular dark field STEM images have been used to visualize nucleic acid molecules positively stained with uranyl acetate. The regular distribution of uranium clusters makes clearly visible the existence of segments of double and single strands on partially denatured RNA molecules. Unpaired regions, as short as about 8 nm, are detectable by this combination of a highly efficient imaging mode and a well adapted preparation technique.

Primary structure of tRNA, rRNA, nRNA, mRNA and viral RNA terminals has been studied. Various RNAs responsible for similar functions have been shown to possess some common features. Previously obtained data on the functional role of RNA terminal sequences, mechanisms of their biosynthesis, the stabilizing role of 5'- and 3'-sequences for the molecules of translated eukaryotic RNAs and some viral RNAs are reviewed.

A significant difference was found to exist in the number of bases per unit length of single-stranded RNA as compared to single-stranded DNA when single-stranded RNA or DNA molecules of known nucleotide sequence were measured by electron microscopy using a cytochrome spreading technique. Using this technique, single-stranded RNA was found to have 17.5% more bases per unit of length than single-stranded DNA. These ratios were verified using four different denaturing conditions for the RNA: 80% formamide, 80% formamide plus glyoxal, 80% formamide/4M urea and 80% formamide/4M urea plus glyoxal. Molecules ranging in size from 1541 to 5386 nucleotides were examined and the number of bases per unit length was found to vary inversely with micrometer was consistent when RNA and DNA molecules of the same length were compared.

Complementary DNA (cDNA) synthesized by Moloney murine sarcoma virus (M-MSV) was separated into two parts, the first, termed MSV-specific cDNA, composed of nucleotide sequences found only in M-MSV viral RNA, and the second, termed MSV-MuLV common cDNA, composed of nucleotide sequences that were found in both M-MSV and murine leukemia virus (MuLV) VIRAL RNAs. RNA complementary to the MSV-specific cDNA was not found in several other MSV isolates, nor in ecotropic MuLV, mouse mammary tumor virus, or several murine xenotropic oncoviruses. Cellular DNA of several species was examined for the presence of nucleotide sequences complementary to MSV-specific cDNA. Cells transformed by M-MSV did contain MSV-specific cDNA in their DNA. Normal mouse cell DNA apparently contained the majority of MSV-specific nucleotide sequences. Cellular DNA of related species contained proportionally less MSV-specific cDNA. Hybrids of MSV-spedivic cDNA and cellular DNA of related species melted at lower temperatures than hybrids of MSV-specific cDNA and mouse cellular DNA. RNA from normal mouse adult or embryonic cells did not contain detectable nucleotide sequences complementary to MSV-specific cDNA. Transformation of cells with M-msv resulted in transcription of RNA hybridizing with MSV-specific cDNA. Methylcholanthrene-induced mouse sarcomas and cell lines derived from them did not contain RNA complementary to MSV-specific cDNA. Mouse cell lines transformed with avian sarcoma virus or Kirsten MSV-specific cDNA. RNA homologous to MSV-specific nucleotide sequences is measurably present only in cells transformed by M-MSV and not in cells transformed by other biological or chemical agents that also cause sarcomas.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.