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PubMed2005

Modular RNA architecture revealed by computational analysis of existing pseudoknots and ribosomal RNAs.

Modular architecture is a hallmark of RNA structures, implying structural, and possibly functional, similarity among existing RNAs. To systematically delineate the existence of smaller topologies within larger structures, we develop and apply an efficient RNA secondary structure comparison algorithm using a newly developed two-dimensional RNA graphical representation. Our survey of similarity among 14 pseudoknots and subtopologies within ribosomal RNAs (rRNAs) uncovers eight pairs of structurally related pseudoknots with non-random sequence matches and reveals modular units in rRNAs. Significantly, three structurally related pseudoknot pairs have functional similarities not previously known: one pair involves the 3' end of brome mosaic virus genomic RNA (PKB134) and the alternative hammerhead ribozyme pseudoknot (PKB173), both of which are replicase templates for viral RNA replication; the second pair involves structural elements for translation initiation and ribosome recruitment found in the viral internal ribosome entry site (PKB223) and the V4 domain of 18S rRNA (PKB205); the third pair involves 18S rRNA (PKB205) and viral tRNA-like pseudoknot (PKB134), which probably recruits ribosomes via structural mimicry and base complementarity. Additionally, we quantify the modularity of 16S and 23S rRNAs by showing that RNA motifs can be constructed from at least 210 building blocks. Interestingly, we find that the 5S rRNA and two tree modules within 16S and 23S rRNAs have similar topologies and tertiary shapes. These modules can be applied to design novel RNA motifs via build-up-like procedures for constructing sequences and folds.

Algorithms↗

PubMedDate not supplied

[Hepatitis delta virus RNA detection by one-step RT-PCR].

We have developed a method of detection HDV RNA where the reverse transcription and amplification are carried out in the same tube, thus reducing the handling time and the contamination risk. RNA extracted from serum or plasma on a microcolumn technique (kit QIAamp viral RNA, QIAgen) is submitted to reverse transcription and amplification by using the One-Step RT-PCR kit (QIAgen). Primers and probe were placed in the conserved ribozymic regions of the HDV genome. The sensitivity of the technique was estimated to 420 copies per reaction by using serial dilutions of a tittered sample. Its specificity was shown by the negativity of 24 samples from anti-HDV negative subjects, either healthy or coinfected by HBV, HCV or HGV. A low tittered sample was reproductively detected in different series. This One-step RT-PCR technique is specific, reproducible and allows the fast detection of active HDV RNA replication. Its adaptation to the routine diagnosis in a laboratory of medical virology is possible and will allow its evaluation on a greater number samples.

Adult↗

PubMed1995

Isolation of a ribozyme with 5'-5' ligase activity.

BACKGROUND: Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from large pools of random-sequence RNAs. We are attempting to use in vitro selection to isolate new ribozymes that have, or can be evolved to have, RNA polymerase-like activities. As phosphorimidazolide-activated nucleosides are extensively used to study non-enzymatic RNA replication, we wished to select for a ribozyme that would accelerate the template-directed ligation of 5'-phosphorimidazolide-activated oligonucleotides. RESULTS: Ribozymes selected to perform the desired template-directed ligation reaction instead ligated themselves to the activated substrate oligonucleotide via their 5'-triphosphate, generating a 5'-5' P1,P4-tetraphosphate linkage. Deletion analysis of one of the selected sequences revealed that a 54-nucleotide RNA retained activity; this small ribozyme folds into a pseudoknot secondary structure with an internal binding site for the substrate oligonucleotide. The ribozyme can also synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages. CONCLUSIONS: The emergence of ribozymes that accelerate an unexpected 5'-5' ligation reaction from a selection designed to yield template-dependent 3'-5' ligases suggests that it may be much easier for RNA to catalyze the synthesis of 5'-5' linkages than 3'-5' linkages. 5'-5' linkages are found in a variety of contexts in present-day biology. The ribozyme-catalyzed synthesis of such linkages raises the possibility that these 5'-5' linkages originated in the biochemistry of the RNA world.

Base Sequence↗

PubMed1997

Characterization of the components and activity of Sonchus yellow net rhabdovirus polymerase.

Sonchus yellow net virus (SYNV) is the best-characterized member of a group of plant rhabdoviruses that replicate in the host cell nucleus. Using a recently developed method for partial purification of active SYNV polymerase by salt extraction of nuclei from infected plant tissue (J. D. O. Wagner et al, J. Virol. 70:468-477, 1996), we have identified the nucleocapsid (N), M2, and L proteins as polymerase complex components (based on copurification with the polymerase activity and by coimmunoprecipitation assays). Furthermore, the L protein was shown by antibody inhibition analysis to be a functional component of the polymerase. A second complex of M2 and L proteins, thought to be a precursor to the polymerase complex, was also identified. In addition, we conducted a detailed characterization of SYNV RNA synthesis in vitro. The results demonstrate that the RNAs are transcribed sequentially, beginning with the N mRNA and followed successively by the remaining five mRNAs in the order of their genome organization. Gene expression conforms to a cascade pattern, with synthesis of the 3'-proximal N mRNA occurring at the highest level, followed by consecutively lower levels of transcription from each subsequent gene. The reaction conditions favor transcription over minus-sense RNA replication, which, we posit, is inhibited near specific signal sequences located on the antigenomic template. The results support the concept that the mechanism of transcription is highly conserved among diverse rhabdoviruses and are compatible with a unified model for the regulation of genomic and antigenomic RNA synthesis.

Antibodies, Viral↗

PubMed1999

Both RNA rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants.

The putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (CLSV) was inserted into an infectious cucumber necrosis tombusvirus (CNV) cDNA clone containing a deletion in the cell-to-cell movement protein gene. Approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric CNV/CLSV cDNA clones developed systemic symptoms 7-19 days postinoculation. Reverse transcription-polymerase chain reaction and sequence analysis of virus obtained from systemically infected leaves indicated that both point mutation and RNA rearrangement (deletion) contributed to the formation of movement competent CNV/CLSV hybrid viruses. The hybrid viruses were found to accumulate to high levels in infected plants, to form stable virions, and to be mechanically transmissible. In addition, a hybrid virus that lacked 50 amino acids at the carboxyl-terminal region of CLSV p27 was still capable of facilitating CNV movement. These data provide experimental evidence for the role of CLSV p27 in viral cell-to-cell movement and demonstrate that p27 can enable efficient movement of the CNV genome. Moreover, the data show that RNA rearrangements known to occur during CNV RNA replication can contribute to rapid evolution of the CNV genome.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and terminal sequence determination of the S and M RNAs of tomato spotted wilt virus.

Complementary DNA to the genomic RNA of tomato spotted wilt virus (TSWV) was synthesized and cloned in either pUC19 or lambda gt10. Restriction endonuclease maps were constructed from cDNA clones specific for the S and the M RNA segments, extending 3.0 and 5.4 kbp respectively. The nucleotide sequences of the 3' and 5' termini of both RNA molecules were determined. The S and M RNAs contain inverted complementary repeats at their termini, probably involved in RNA replication and in the formation of circular nucleocapsids in virions. The terminal structures of the TSWV genome resemble, in these aspects, those of the Bunyaviridae.

Base Sequence↗

PubMed2000

E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies.

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.

Animals↗

PubMed2004

Application of the trak-C HCV core assay for monitoring antiviral activity in HCV replication systems.

The Ortho trak-C immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5pg, and exhibits linear detection up to 100pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-alpha using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C assay has several advantages over RNA-based methods of viral monitoring.

Animals↗

PubMed1986

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.

Capsid↗

PubMed1990

Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line.

A trimer of hepatitis delta virus (HDV) cDNA in a retrovirus expression vector was transfected into subclone of the PLC/PRF/5 human hepatoma cell line, and a stable cell line (H1 delta 9) was clonally selected that supported the synthesis of both genomic and antigenomic sense HDV RNA. The H1 delta 9 cell line also expressed hepatitis delta antigen (HDAg) in cell nuclei in three distinct morphological patterns, including patterns typically seen in HDV-infected livers. HDAg expression was restricted to the smaller (p24) of the two HDAg-associated polypeptides in early passages of the H1 delta 9 cell line, but continuous passage of the cells resulted in increasing of expression of the larger (p27) HDAg-specific polypeptide. Passage of the H1 delta 9 cells also led to sustained expression of monomeric HDV RNA and a reduction in the levels of dimeric- and trimeric-HDV RNA. This was accompanied by an attenuation of virus-related cytotoxicity which was a feature of early cell passage numbers. HDV RNA replication in these cells was resistant to actinomycin D suggesting that replication was not dependent on continued expression from the transfected HDV cDNA and thus was likely to be self-sustaining.

Blotting, Northern↗

PubMed1991

Identification of RNA replicase subunits responsible for initiation of RNA synthesis of tick-borne encephalitis virus by affinity labelling.

Porcine embryo kidney cells infected by tick-borne encephalitis virus (TBEV) were fractionated into nuclear, membrane, and cytoplasmic fractions. To identify proteins involved in the initiation of RNA replication at different stages of infection a highly specific affinity labelling technique was used. In samples of the nuclear fraction taken from cells 45 h after infection (late stage), affinity labelling with aldehyde-containing derivatives of ATP and elongation of this label with [alpha-32P]GTP identified a polypeptide with a molecular mass of about 69 kDa. By means of affinity labelling with aldehyde-containing analogues of GMP, GDP, and GTP as initiation substrates and [alpha-32P]ATP as the elongation substrate, a polypeptide of 100 kDa was selectively modified in the nuclear fraction of cells at the early stages of infection (8 h). These proteins were immunostained with TBEV-specific antibodies, and were identified as the nonstructural TBEV proteins NS3 and NS5, respectively. It was concluded that NS3 and NS5 take part in the initiation of TBEV genome replication at the late and early stages of infection, respectively.

Animals↗

PubMed1997

Detection of RNA sequences in cultures of a stealth virus isolated from the cerebrospinal fluid of a health care worker with chronic fatigue syndrome. Case report.

A cytopathic stealth virus was cultured from the cerebrospinal fluid of a nurse with chronic fatigue syndrome. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the patient's culture yielded positive results with primer sets based on sequences of a previously isolated African green monkey simian-cytomegalovirus-derived stealth virus. The same primer sets did not yield PCR products when tested directly on DNA extracted from the cultures. The findings lend support to the possibility of replicative RNA forms of certain stealth viruses and have important implications concerning the choice of therapy in this type of patient.

Adult↗

PubMed1997

Recognition of bacteriophage Qbeta plus strand RNA as a template by Qbeta replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor.

RNA-protein interactions between bacteriophage Qbeta plus strand RNA and the components of the Qbeta replicase system were studied by deletion analysis. Internal, 5'-terminal and 3'-terminal deletions were assayed for template activity with replicase in vitro. Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity. Binding complexes of the deletion mutant RNAs with the different proteins involved in Qbeta RNA replication were analysed by electron microscopy. The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site. The same types of complexes observed with replicase were also formed with purified protein S1 (the alpha subunit of replicase), suggesting that these internal interactions with Qbeta RNA are mediated by the S1 protein. The Qbeta host factor, a protein required for the template activity of the Qbeta plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3'-end, resulting in double-looped structures. The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites. An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs. Qbeta RNA molecules with 3'-truncations formed 3'-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3'-end by host factor is not dependent on a specific 3'-terminal base sequence.

Allolevivirus↗

PubMed2003

The R protein of SARS-CoV: analyses of structure and function based on four complete genome sequences of isolates BJ01-BJ04.

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.

Amino Acid Sequence↗

PubMed2004

The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation.

The bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase can initiate RNA replication by a de novo mechanism without a primer. The structure of BVDV polymerase, determined to 2.9-A resolution, contains a unique N-terminal domain, in addition to the fingers, palm, and thumb domains common to other polymerases. The structure of BVDV polymerase complexed with GTP, which is required for de novo (primer-independent) initiation, shows that GTP binds adjacent to the initiation NTP, suggesting that the GTP mimics a vestigial RNA product. Comparison of five monomers in two different crystal forms showed conformational changes in the fingertip region and in the thumb domain that may help to translocate the RNA template and product strands during elongation. The putative binding sites of previously reported BVDV inhibitors are also discussed.

Amino Acid Sequence↗

PubMed1991

Sequence analysis of the S RNA of the African arenavirus Mopeia: an unusual secondary structure feature in the intergenic region.

Mopeia virus is an apparently nonpathogenic African arenavirus which can protect animals from subsequent challenge by the closely related Lassa virus. As a step toward understanding these differences in pathogenicity and the means by which Mopeia virus infection can protect against subsequent Lassa virus infection, cDNA clones corresponding to 3419 nucleotides of Mopeia virus S RNA were isolated and sequenced. Two open reading frames, encoding the glycoprotein precursor (GPC) and nucleocapsid (N) proteins, were located in the ambisense arrangement characteristic of the arenaviruses. Comparison of the amino acid sequences of the translation products with those of two Lassa virus strains showed considerable conservation, with 74 and 80% identity for the two glycoproteins G1 and G2, and 74% identity for the N protein. The putative dibasic site of GPC cleavage (R-R) was conserved, as were the potential N-linked glycosylation sites. A striking difference between Mopeia virus and Lassa virus was identified in the noncoding intergenic region. Instead of the single hairpin structure formed by base-pairing of complementary sequences which is usually found, the Mopeia virus S RNA has the potential to form two hairpins. These hairpins were similar in sequence and may have been formed in a duplication event during RNA replication. The possible contribution of this secondary structure feature to differences in pathogenicity between Mopeia and Lassa viruses is discussed.

Amino Acid Sequence↗

PubMed1997

Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse.

The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.

Administration, Intranasal↗

PubMed2004

Cis and trans requirements for rolling circle replication of a satellite RNA.

Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.

Avena↗