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At least 1,369 records · Page 76Linked to original sources
PubMed2005

The flavivirus-conserved penta-nucleotide in the 3' stem-loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation.

A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3' untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3' UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5'-CACAG-3' located at the top of the 3' stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication.

Conserved Sequence↗
PubMed2005

Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA.

Satellite (sat) RNAs are parasitic sub-viral RNA replicons found associated with certain positive-strand RNA viruses. Typical sat RNAs, such as those associated with members of the genus Tombusvirus, share little or no sequence identity with their helper virus genomes. Here, we have investigated a tombusvirus sat RNA and determined that it contains two functionally-relevant higher-order RNA domains, a T-shaped domain and a downstream domain, that are similar to elements shown previously to be present in the 5' untranslated regions (UTRs) of tombusvirus genomes. Although the two sat RNA domains showed only limited sequence identity with their viral counterparts, they were able to adopt comparably-folded RNA secondary structures. Interestingly, the relative spacing between the domains in the viral and satellite contexts was notably different. In the viral 5' UTR, the two domains are adjacent and separated by a small hairpin, however, in the sat RNA they are separated by a 137-nt long segment. Despite this distal modular arrangement, the two domains were found to be united spatially in the sat RNA through the formation of an RNA-RNA bridge. This co-localization facilitated an important inter-domain interaction and was essential for efficient helper-mediated sat RNA accumulation in protoplasts. These results indicate that the tombusvirus sat RNA and helper genome contain structurally and functionally equivalent RNA domains. It is proposed that the limited sequence identity observed between these corresponding higher-order RNA structures is related to a strategy that reduces the induction of gene silencing, which presumably would be detrimental to both viral and sat RNA replicons.

Base Sequence↗
PubMed2005

Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Beta vulgaris↗
PubMed2006

A mouse cell-adapted NS4B mutation attenuates West Nile virus RNA synthesis.

An adaptive mutation (E249G) within West Nile virus (WNV) NS4B gene was consistently recovered from replicon RNAs in C3H/He mouse cells. The E249G is located at the C-terminal tail of NS4B predicted to be on the cytoplasmic side of the endoplasmic reticulum membrane. The E249G substitution reduced replicon RNA synthesis. Compared with the wild-type NS4B, the E249G mutant protein exhibited a similar efficiency in evasion of interferon-beta response. Recombinant E249G virus exhibited smaller plaques, slower growth kinetics, and lower RNA synthesis than the wild-type virus in a host-dependent manner, with the greatest difference in rodent cells (C3H/He and BHK-21) and the least difference in mosquito cells (C3/36). Selection of revertants of E249G virus identified a second site mutation at residue 246, which could compensate for the low replication phenotype in cell culture. These results demonstrate that distinct residues within the C-terminal tail of flavivirus NS4B are critical for viral replication.

Adaptation, Physiological↗
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↗
PubMed1999

Biological consequences of deletions within the 3'-untranslated region of flaviviruses may be due to rearrangements of RNA secondary structure.

It was previously reported that deletions introduced into the 3'-untranslated region (3'-UTR) of dengue type 4 (DEN 4) virus (Men, R., Bray, M., Clark, D., Chanock, R.M., Lai, C.J., 1996. DEN 4 virus mutants containing deletions in the 3'-noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in Rhesus monkeys. J. Virol. 70, 3930-3937), tick-borne encephalitis (TBE) virus (Mandl, C.W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P.F., Allison, S.L. , Heinz, F.X., 1998. Spontaneous and engineered deletions in the 3'-noncoding region of TBE virus: construction of highly attenuated mutants of a flavivirus. J. Virol. 72, 2132-2140) and subgenomic replicons of Kunjin virus (Khromykh, A.A., Westaway, E.G., 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 71, 1497-1505) altered the infectivity of the mutants and reduced the efficiency of RNA replication. Here, these deletions were superimposed onto the models of secondary structure we constructed previously and the folding of the modified 3'-UTR sequences was simulated. The analysis showed that most of the deletions disrupted or reshaped conserved elements of secondary structure and that the biological effects of these deletions are likely to represent structural rearrangements in the 3'-UTR, rather than the loss of sequence motifs. The analysis also suggested that the overall structural integrity of the flaviviral 3'-UTR is essential for optimal performance of its promotor function, although two distinct parts can be defined: the most 3'-terminal structures and sequences which may be critical for the initiation of minus-strand RNA synthesis, and more proximal structures and sequences that possibly function as enhancers of viral RNA replication. The functional significance of certain structural elements and their possible effect on the efficiency of viral replication in different cells are also discussed.

3' Untranslated Regions↗
PubMed2001

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Animals↗
PubMed2003

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis.

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.

Archaeal Proteins↗
PubMed1996

Plasmid vectors for gram-positive bacteria switching from high to low copy number.

A set of vectors for Gram-positive bacteria was constructed with a new feature which enables the switching down of their copy number per cell. These vectors carry the replication region of pAM beta 1, containing a gene essential for replication, repE, and its regulator, copF. The latter gene was inactivated by inserting a linker into its unique KpnI site. Since copF downregulates the expression of repE, its inactivation leads to an increase in the plasmid copy number per cell. The original low copy state can be restored by removal of the linker via KpnI cleavage and ligation. The new replicon was used to build (i) vectors for studying gene regulation by transcriptional or translational fusion with the bacterial luciferase gene, (ii) vectors for gene expression, and (iii) cassettes of the replicon with different multiple cloning sites, which would facilitate construction of vectors for novel purposes.

Amino Acid Sequence↗
PubMed1998

TrfA dimers play a role in copy-number control of RK2 replication.

Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of 'handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication. However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules.

Bacterial Proteins↗
PubMed1998

Quasispecies and the implications for virus persistence and escape.

BACKGROUND: In the 1970s Manfred Eigen and colleagues proposed a new model of molecular evolution to explain adaptability and rapid evolution of simple replicons, as those that probably populated the earth at the onset of life. This model of evolution placed emphasis on mutant generation, to the point of invalidating the concept of wild-type genomes as a defined sequence of nucleotides. In striking similarity with the proposals for such early replicons, present-day RNA viruses consist of complex distributions of nonidentical but closely related genomes termed quasispecies. OBJECTIVES: To discuss indeterminations inherent to a quasispecies structure and to the analytical procedures to define it, biological implications of quasispecies, and the need to take into account this type of population structure, in order to design effective strategies to prevent and control diseases caused by highly variable viruses. RESULTS: Quasispecies have many biological implications, extending from viral pathogenesis to the emergence of new pathogens, rapid antigenic variation, and alterations in cell tropism, virulence, host range and viral gene expression. CONCLUSIONS: Diseases caused by highly variable RNA viruses prove very difficult to control and vaccine development against such viruses are largely unsuccessful. It is important to understand quasispecies composition and dynamics, as quasispecies are an important step in the natural history of RNA viruses.

Animals↗
PubMed1983

Chromatin structural changes in synchronized cells blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade.

We have investigated the loss of histone H1 from chromatin [D'Anna, J. A., Gurley, L. R., & Tobey, R. A. (1982) Biochemistry 21, 3991-4001] and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blockade. Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block. As H1 is lost, chromatin appears to undergo structural change. After 10 h of HU block, the new deoxyribonucleic acid (DNA) and a portion of the old DNA have measured nucleosome repeat lengths (37 degrees C digestion) which are less than those of controls and similar to those observed by Annunziato and Seale [Annunziato, A. T., & Seale, R. L. (1982) Biochemistry 21, 5431-5438] for new immature chromatin in the absence of HU. By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease. Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that (1) the distribution of mononucleosome species changes during HU block and (2) some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels. The results raise the possibility that (1) during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and (2) H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until the replication fork has passed and, perhaps, a substantial portion of the replicon has been replicated.

Animals↗
PubMed1984

Changes in histone H1 content and chromatin structure of cells blocked in early S phase by 5-fluorodeoxyuridine and aphidicolin.

We have measured changes in histone H1 content and changes in chromatin structure of Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation and blockade with 5-fluorodeoxyuridine or aphidicolin. Both the H1:core histone ratio in isolated nuclei and the H1 content of the cell are reduced 20-60%, depending on the duration of the block. The new deoxyribonucleic acid (DNA) synthesized during S-phase block has a shorter nucleosome repeat length than that of bulk chromatin, but it is nearly equally resistant as bulk DNA to attack by micrococcal nuclease. During the time that H1 content is decreasing, bulk chromatin also undergoes structural changes so that its nucleosome cores appear to be more closely packed along the DNA chain. The losses in H1 content and changes in chromatin structure are similar to those reported for cells blocked in early S phase by hydroxyurea [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. The results suggest that losses of H1 and changes in chromatin structure are general events which occur when the elongation of initiated replicons or the joining of intermediate-sized DNA fragments is retarded during replication. They are consistent with the notions that H1 is lost from initiated replicons and/or the loss of H1 is part of an alarm response in the cell which might facilitate events leading to gene amplification.

Animals↗
PubMed2005

Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication.

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.

Antiviral Agents↗
PubMed2001

Characterization of RNA synthesis and translation of bovine viral diarrhea virus (BVDV).

Full length and replicon genomes of various strains of bovine viral diarrhea virus (BVDV) have been characterized. Analysis of growth kinetics for a pair of cytopathogenic (cp) and noncytopathogenic (ncp) strains revealed that ncp strain synthesized viral RNA at much reduced level compared to the cp strain. Kinetics of translation and replication, the effects of bi-cistronic versus mono-cistronic genomes, and cis requirements for viral replication were also examined in a BVDV replicon D19c. Importantly, our results suggest a tight regulation and a switch from translation to replication, and demonstrated the cis requirements of NS4B and NS5A in replication.

Diarrhea Viruses, Bovine Viral↗
PubMed2001

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

Journal Article↗
PubMed2004

Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea.

The pNB101 is the first plasmid to be isolated from an haloalkaliphilic archaea. With insertion of the ColE1 replicon of Escherichia coli, as well as two antibiotic resistance genes at its unique Hin dIII site, a novel shuttle vector between haloarchaea and E. coli was developed. This vector, named pNB102, was successfully transformed into two non-alkaliphilic haloarchaea, Halobacterium salinarum SNOB and Haloarcula hispanica ATCC33960. The presence and stability of pNB102 in the transformants were confirmed by PCR identification, Southern blotting and restriction endonuclease digestion. Results also indicated that the presence of restriction-modification (R-M) systems in some Halobacterium species prevented this transformation. It is the first report that the replicon of pNB101 has such a wide host range, and has taken the first step for construction of the vector/host system in haloalkaliphilic archaea.

Archaea↗