Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.     

Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae

The (+) single-stranded RNA (ssRNA) of the L-A virus is the species packaged to form new viral particles. Empty L-A viral particles specifically bind viral (+) ssRNA, and a sequence 400 bases from the 3' end is necessary for this activity. We show that its stem-loop structure, the A residue protruding from the stem, and the loop sequence are all important for the binding, and that this 34 base region is sufficient for the binding. M1, a satellite virus of L-A, has a similar structure on its (+) strand that is likewise sufficient for the binding. Heterologous RNA with the binding sequence from L-A or M1, when expressed in vivo, was packaged in L-A viral particles. Thus, the sites necessary to bind to empty particles are encapsidation signals for the L-A virus. Since the pol domain of the 180 kd minor coat protein appears to be responsible for the binding, this result suggests that the RNA polymerase molecule recognizes the viral genome for packaging.     

Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.     

An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.     

Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).     

The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells.

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).     

Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.     

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

Identification of a Plant Viral RNA Genome in the Nucleus

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.     

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.