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At least 919 records · Page 51Linked to original sources
PubMed1990

Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II.

Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation. The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies. This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II. In these complexes, most loops of RNA II interact with their complementary loops of RNA I. The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes. Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product.

Bacteriocin Plasmids↗
PubMed1970

Effect of mengovirus replication on choline metabolism and membrane formation in novikoff hepatoma cells.

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.

Animals↗
PubMed2004

Advances in the molecular biology of tombusviruses: gene expression, genome replication, and recombination.

The tombusviruses are among the most extensively studied messenger-sensed RNA plant viruses. Over the past decade, there have been numerous important advances in our understanding of the molecular biology of members in this genus. Unlike most other RNA viruses, the synthesis of tombusvirus proteins has been found to involve an atypical translational mechanism related to the uncapped and nonpolyadenylated nature of their genomes. Tombusviruses also appear to employ an unusual mechanism for transcription of the sg mRNAs that template translation of a subset of their viral proteins. In addition to these new insights into tombusvirus gene expression, there has also been significant progress made in our understanding of tombusvirus RNA replication. These studies have been facilitated greatly by small genome-derived RNA replicons, referred to as defective interfering RNAs. In addition, the development of an in vitro system to study viral RNA synthesis has allowed for dissection of some of the steps involved in the replication process. Another exciting recent advance has been the creation of yeast-based systems that support amplification of tombusvirus RNA replicons and will allow the identification of host factors involved in viral RNA synthesis. Lastly, the recombinogenic nature of tombusvirus genomes has made them ideal systems for studying RNA-RNA recombination and genetic rearrangements, both in vivo and in vitro. In this review, we compile recent information on each of the aforementioned processes-translation, transcription, replication and recombination-and discuss the significance of the results.

Base Sequence↗
PubMed2001

Hepatitis C viral RNA: challenges and promises.

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus of the Flaviviridae family, is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Its RNA is difficult to study because biological materials are scarce and RNA replication is of low efficiency. This review focuses on the structure and functions of HCV RNA along with their biological and clinical significance. Despite the challenging characteristics of HCV, significant progress has been made in understanding the properties of HCV RNA and developing viral replication systems toward the improvement of antiviral therapies.

Base Sequence↗
PubMed2002

Co-evolution of the genetic code and ribozyme replication.

The origin of translation has stimulated much discussion since the basic processes involved were deciphered during the 1960s and 1970s. One strand of thought suggested that the process originated from RNA replication in the RNA world (Weiner & Maizels, 1987, 1994). In this paper I seek to extend this model. The mRNA originates as a replication intermediate of minus-strand ribozyme replication and thus contains all the genetic information contained in both the ribozyme portion and the putative tRNA-like portion of the RNA molecule. Qualitatively, this is similar to the model for the origin of chromosomes (Szathmary & Maynard-Smith, 1993, Maynard-Smith & Szathmary, 1993). This model explicitly describes the evolution of early chromosomes and the role replication played in generating the modern mRNA. Moreover, by pursuing this model, the START and STOP codons were derived and their original function with regard to the primitive 23S ribosomal RNA is suggested. Co-evolution of the genetic code (Wong, 1975) is also contained within the model. Lastly, I address some of the benefits and costs that the process may have for the organism in the context of autotrophy in the RNA world.

Animals↗
PubMed2004

Novel insights into hepatitis C virus replication and persistence.

Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the family Flaviviridae. A hallmark of HCV is its high propensity to establish a persistent infection that in many cases leads to chronic liver disease. Molecular studies of the virus became possible with the first successful cloning of its genome in 1989. Since then, the genomic organization has been delineated, and viral proteins have been studied in some detail. In 1999, an efficient cell culture system became available that recapitulates the intracellular part of the HCV life cycle, thereby allowing detailed molecular studies of various aspects of viral RNA replication and persistence. This chapter attempts to summarize the current state of knowledge in these most actively worked on fields of HCV research.

Genome, Viral↗
PubMed1997

A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.

Animals↗
PubMed2001

Oligomeric structures of poliovirus polymerase are important for function.

Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

Amino Acid Substitution↗
PubMed2005

Subversion of cellular autophagosomal machinery by RNA viruses.

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

Autophagy↗
PubMed1989

The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.

Capsid↗
PubMed2026

EV-B 3D polymerase remodels viral populations through 5'UTR recombination to subvert cardiac antiviral innate immunity.

Viral myocarditis, a leading cause of morbidity in young populations, is strongly linked to Coxsackievirus B (CV-B) infections harboring dominant 5'-terminally deleted (5'TD) and minor full-length (FL) CV-B RNA populations in cardiac tissues. Here, we demonstrate how viral RNA-dependent RNA polymerase (3Dpol)-driven recombination in the 5'UTR orchestrates viral RNA populations dynamics and subverts type I interferon responses. In primary human cardiomyocytes (HCMs), 3Dpol-mediated copy-choice recombination enhances 5'TD RNA replication while suppressing FL populations. Infection of immunocompetent mice with recombination-deficient CV-B3 (3Dpol Y276H) shifted 5'TD populations ratios toward immune-sensing viral RNAs, elevating cardiac IFN-β/ISG15 and accelerating viral clearance. Transfection experiments confirmed that 50-nt 5'TD RNAs (TD50) evade innate immunity, whereas shorter deletions (9-36-nt, TD15) restore type I interferon responses in HCMs. Our findings establish 3Dpol-driven recombination as a critical mechanism sustaining pathogenic 5'TD RNAs that subvert antiviral innate immunity, highlighting recombination inhibition as a promising therapeutic strategy against CV-B myocarditis.

Animals↗
PubMed2001

Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants.

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01(+) rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01(+) monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.

Animals↗
PubMed1998

Targets for inhibition of hepatitis C virus replication.

Considerable progress has been made in characterizing the proteins involved in hepatitis C virus (HCV) replication, despite the lack of a cell culture system. A number of systems have been developed to examine the processes involved in viral replication, including the initiation and processing of the viral proteins required for RNA replication, the unwinding activities of the RNA helicase and the synthesis of RNA by the viral polymerase. These processes have been examined using individually cloned proteins expressed in various in vitro systems, which may be suitable targets for antiviral agents. The viral helicase and protease domains have now been crystallized, which may enable the rational design of specific inhibitors. The recent developments in HCV research in understanding the function of the viral non-structural proteins and the establishment of in vitro screening assays may aid in the development of new antiviral agents.

Antiviral Agents↗
PubMed2000

The leader RNA of coronavirus mouse hepatitis virus contains an enhancer-like element for subgenomic mRNA transcription.

While the 5' cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize the cis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of the cis-acting signal for replication in the 5' untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5'-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5' untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5' cis-acting sequence regulates subgenomic RNA transcription. The 5'-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines the cis-acting transcription signal at the 5' end of the DI genome.

5' Untranslated Regions↗
PubMed1990

Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.

Base Sequence↗
PubMed1998

The middle subunit of replication protein A contacts growing RNA-DNA primers in replicating simian virus 40 chromosomes.

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, its size distribution, its RNA primer content, and its replication fork polarity were determined [corrected]. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase alpha-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.

Cross-Linking Reagents↗