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At least 721 records · Page 40Linked to original sources
PubMed2005

A hypothetical pathway from the RNA to the DNA world.

If the DNA world was preceded by a RNA world as widely suggested a rational pathway should be discernable to link the two. This report uses as a starting point a membrane-enclosed ribozyme capable of polymerising itself and its counterpart copy. As molecular complexity increased, it is suggested that a consortia of the initial ribozyme polymerase and chaperone molecules formed a complex specifically for RNA replication. A mutation in one of several copy-genomes coding for these replication machines then led in step-wise fashion to a proto-ribosome that increasingly inserted specific amino acids instead of nucleotides into a growing RNA chain, the driving force being selection for improved or new function. Eventually the nucleotides would be entirely displaced in this proto-ribosome, after which the ribose-phosphate linkage would be replaced by peptide linkage. The final steps would be the formation of DNA from the RNA genomic material via reverse transcriptase, coupled with the evolution of enzymes for DNA polymerisation and transcription. At this point the original RNA-replicator machinery would be redundant and eliminated, the RNA genomic material would become mRNA and the present-day function of the ribosome would be fixed. In the scenario described a mechanism for the selection for L: -amino acids becomes evident.

Amino Acids↗
PubMed2003

3' nontranslated RNA signals required for replication of hepatitis C virus RNA.

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.

3' Untranslated Regions↗
PubMed1998

Involvement of antisense RNA in replication control of the lactococcal plasmid pND324.

pND324 belongs to a family of closely related theta-type plasmids from Lactococcus lactis. An antisense RNA, termed countertranscript (ctRNA), was identified which is complementary to the leader sequence of the mRNA that encodes RepB, a protein essential for plasmid replication. When the synthesis of ctRNA was abolished by site-directed mutagenesis within its promoter region, the mutant replicon showed a 1.8-fold increase in copy number. Similar ctRNA promoter sequences are readily identifiable in 12 other published lactococcal theta-type plasmids, suggesting that they all encode a similar ctRNA-mediated regulatory mechanism.

Base Sequence↗
PubMedDate not supplied

Template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added template, standard preparations of Q beta replicase spontaneously synthesize RNA in vitro, possibly as a result of RNA contamination. Using special enzyme purifications, Sumper and Luce presented evidence that self-replicating RNA not present ab initio can grow out of 'template-free' incorporation mixtures. In contrast to DNA polymerase I and RNA polymerase, which also show de novo synthesis, the products synthesized 'de novo' by Q beta replicase are RNA species containing nonrepetitive sequences of defined lengths which differ between experiments, even when synthesized under identical conditions, in fingerprints, chain lengths and kinetic parameters. Kinetic analysis of the de novo processes distinguished it from template-instructed synthesis and excluded an assumption of self-replicating RNA contamination. These conclusions were questioned recently by Hill and Blumenthal, who claimed to show that highly purified Q beta replicase preparations cannot produce RNA de novo. We now present evidence that, under the conditions required for de novo synthesis, Q beta replicase prepared according to their method is also capable of de novo synthesis. Furthermore, we show that Q beta replicase condenses nucleoside triphosphates to more or less random oligonucleotides.

Base Sequence↗
PubMed1983

Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains.

Mouse hepatitis virus A59 codes for seven mRNAs in infected cells. These mRNAs are transcribed from a minus (-) strand template of genome length and contain a leader RNA at their 5' ends. To further elucidate the mechanism of coronavirus transcription, we examined the structure of mouse hepatitis virus replicative intermediates (RIs) isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography. Purified RIs migrated as a single species on agarose gels and sedimented between 12 and 38S on 10 to 25% sucrose gradients. The complexes were readily heat denatured into a heterogeneous population of smaller RNA molecules which probably represent nascent plus (+) strands. RNase A digestion of RIs produced a single replicative form which sedimented between 30 and 32S. These data suggest that the RI is composed of a single genome-sized (-) strand hydrogen bonded to an average of 4 to 6.5 nascent (+) strands. In contrast, a column-purified replicative form was extremely resistant to RNase A digestion and heat denaturation and migrated as a single RNA species on agarose gels and sucrose gradients. Oligonucleotide fingerprinting of an RI revealed the presence of the 5' leader RNA on the nascent (+) strands. In addition, an average of 6.2 cap structures were present in each RI, which agrees with the average number of nascent (+) strands per RI. These data suggest that the leader RNA is utilized as a primer for mouse hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.

Animals↗
PubMed2002

A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs.

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA-->cRNA-->vRNA), but its transcriptional activity (vRNA-->mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.

Amino Acid Sequence↗
PubMed2001

Identification of sequences in rotavirus mRNAs important for minus strand synthesis using antisense oligonucleotides.

The core of the rotavirion consists of three proteins, including the viral RNA polymerase, and 11 segments of double-stranded (ds)RNA. The RNA polymerase of disrupted (open) cores is able to catalyze the synthesis of dsRNA from exogenous viral mRNAs in vitro. In this study, we have identified sequences in exogenous viral mRNAs important for RNA replication using antisense oligonucleotides. The results showed that oligonucleotides complementary to the highly conserved 3'-terminal sequence of rotavirus mRNAs prevented all but basal levels of dsRNA synthesis. Notably, we observed that the addition of oligonucleotides which were complementary to nonconserved sequences present either at the 5'- or 3'-end of a viral mRNA effectively inhibited its replication without interfering with the replication of other viral mRNAs present in the same replication assay. Thus, the nonconserved sequences in rotavirus mRNAs contain gene-specific information that promotes RNA replication. The fact that antisense oligonucleotides inhibited dsRNA synthesis indicates that the strandedness (single- versus double-stranded) and secondary structure of the viral mRNA template are factors that affect the efficiency of minus strand synthesis.

3' Untranslated Regions↗
PubMed1998

Inhibition of Cellular RNA polymerase II transcription by delta antigen of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA and the hepatitis delta antigen (HDAg) protein. The viral RNA is replicated via RNA-dependent RNA synthesis, which is thought to be mediated by host DNA-dependent RNA polymerase II (pol II). The precise mechanism of HDV RNA replication using RNA as a template remains to be elucidated, though it is clear that HDAg is involved. We demonstrate here that both SP1-activated and basal pol II transcription are inhibited by HDAg. This inhibitory effect of HDAg was observed in vivo in transient cotransfection assays as well as in vitro in HeLa nuclear extracts with purified, recombinant HDAg. The in vitro inhibition of pol II transcription could be reversed with excess HeLa nuclear extracts. Furthermore, HDAg specifically inhibited pol II-mediated transcription but not pol I- or III-mediated transcription. These results provide support for the model in which HDAg participates in a complex with host cell pol II transcription factors to mediate pol II-dependent HDV RNA replication, concomitantly cellular pol II transcription.

Chloramphenicol O-Acetyltransferase↗
PubMed2005

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses.

Viruses are devastating pathogens of humans, animals, and plants. To further our understanding of how viruses use the resources of infected cells, we systematically tested the yeast single-gene-knockout library for the effect of each host gene on the replication of tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins, and other compounds and in protein targeting/transport. Comparison with published genome-wide screens reveals that the replication of TBSV and brome mosaic virus (BMV), which belongs to a different supergroup among plus-strand RNA viruses, is affected by vastly different yeast genes. Moreover, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related alpha-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This finding suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may suggest connections between chronic diseases and prior virus infection.

Blotting, Northern↗
PubMed1975

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.

Adenosine Triphosphate↗
PubMed2006

Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging.

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Animals↗
PubMed2003

Cell-free synthesis of encephalomyocarditis virus.

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.

Animals↗