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At least 1,261 records · Page 70Linked to original sources

PubMed1996

Mosquito homolog of the La autoantigen binds to Sindbis virus RNA.

We have isolated a 50-kDa mosquito protein that binds with high affinity to a riboprobe representing the 3' end of the minus strand of Sindbis virus RNA. The isolated protein has been used to obtain cDNA clones encoding this protein that have been sequenced and used to express the protein in large amounts. Sequence comparisons make clear that this protein is the mosquito homolog of the La autoantigen. The N-terminal half of the protein shares considerable sequence identity with the human La protein, the rat La protein, and the recently identified Drosophila melanogaster homolog. There is one stretch of 100 amino acids in the N-terminal domain in which 48 residues are identical in all four proteins. In contrast, the C-terminal domain of the mosquito protein shares little identity with any of the other three proteins. We have also shown that the mosquito protein, the human protein, and a putative chicken homolog of the La protein cross-react immunologically and, thus, all share antigenic epitopes. The mosquito La protein is primarily nuclear in location, but significant amounts are present in the cytoplasm, as is the case for the La proteins of other species. The equilibrium constant for the binding of the expressed mosquito La protein to the Sindbis virus riboprobe is 15.4 nM, and thus the affinity of binding is high enough to be physiologically relevant. Furthermore, the conservation of this protein in the animal kingdom may be significant, because Sindbis virus utilizes mosquitoes, birds, and mammals as hosts. We propose that the interactions we observe between the La protein and toes, birds, and mammals as hosts. We propose that the interactions we observe between the La protein and a putative promoter in the Sindbis virus genome are significant for Sindbis virus RNA replication.

Aedes↗

PubMed1994

Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells.

Poliovirus (PV)-infected cells undergo extensive proliferation and rearrangement of intracellular smooth membranes to generate vesicles on which viral RNA replication occurs. PV proteins 2C and 2BC are known to be tightly associated with these membranous replication complexes and have been proposed to be involved in the formation of these virus-induced vesicles. We have expressed these proteins, and proteins with mutations in the putative nucleotide (NTP) binding motifs, in human cells using recombinant vaccinia viruses and T7 RNA polymerase-directed transcription. To ascertain the subcellular localization properties of these proteins in the absence of other PV proteins and to determine whether they induced ultrastructural changes, cells expressing 2C and 2BC proteins were examined by immunofluorescence (IF) microscopy, electron microscopy (EM), and immuno-EM (IEM). The cytoplasm of cells expressing either 2C or 2BC exhibited vesicles of 50-350 nm in diameter, which resembled those found in PV-infected cells. Both 2C and 2BC were associated with these vesicles. Mutations in the putative NTP binding motif did not affect vesicle induction by 2C or 2BC. Despite the membrane reorganization and vesicle formation induced by 2C and 2BC proteins, no enhanced synthesis of lipid was observed. Guanidine hydrochloride at a concentration that inhibits PV replication, did not have significant effects on the IF patterns of either 2C or 2BC. An additional prominent alteration in cells expressing 2C, but not 2BC, was the formation of extensive tubular membrane structures with a myelin-like arrangement in the lumen of the rough endoplasmic reticulum. IEM analyses showed that 2C was associated with these structures. In the presence of other PV proteins, the tubular membrane structures induced by 2C were not detected. These structures are not observed in poliovirus-infected cells, but likely indicate a novel property of 2C that induces a complex interaction with intracellular membranes.

Amino Acid Sequence↗

PubMed2006

Cell-type-specific growth restriction of vesicular stomatitis virus polR mutants is linked to defective viral polymerase function.

Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

Animals↗

PubMed2003

Mechanism of action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTP-binding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.

Allosteric Site↗

PubMed2002

The 3'-untranslated region of RNA1 as a primary determinant of temperature sensitivity of Red clover necrotic mosaic virus Canadian strain.

Red clover necrotic mosaic virus Canadian strain (RCNMV-Can) induces symptoms on host plants at 17 degrees C, but not at 25 degrees C. We investigated the temperature sensitivity of RCNMV-Can in Nicotiana benthamiana plants and protoplasts using infectious transcripts of genomic RNAs 1 and 2. Viral RNAs accumulated in both inoculated and noninoculated leaves at 17 degrees C, whereas no viral RNAs were detected at 25 degrees C in either inoculated or noninoculated leaves. Similar temperature sensitivity in RNA accumulation was observed in protoplasts, and no viral RNAs were detected at temperatures above 22 degrees C. These results indicate that the temperature sensitivity of RCNMV-Can occurs at an early stage of infection, including during RNA replication. Using reassortant viruses and chimeric RNAs 1 between RCNMV-Can and the RCNMV Australian strain, which accumulates viral RNAs at nonpermissive temperatures for RCNMV-Can, we demonstrated that a viral determinant for the temperature sensitivity resides in the 3'-untranslated region of RNA1.

3' Untranslated Regions↗

PubMed1980

The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.

The 3'- and 5'-terminal nucleotides of the genome segments of an influenza A, B, and C virus were identified by directly sequencing viral RNA using two different sequencing techniques. A high degree of conservation at the 3' ends as well as at the 5' ends was observed among the genome segments of each virus and among the segments of the three different virus types. A uridine-rich region was observed from positions 17 through 22 at the 5' end of each segment. Moreover, the conserved 3' and 5'-terminal sequences showed partial and inverted complementarity. This feature results in very similar sequences at the 3' ends of the plus and minus strand RNAs and may also enable single-strand RNAs of influenza virus to form "panhandle" structures. Inverted complementary repeats may play an important role in initiation of viral RNA replication.

Base Sequence↗

PubMed1999

Tertiary structure stabilization promotes hairpin ribozyme ligation.

The hairpin ribozyme catalyzes a reversible RNA cleavage reaction that participates in processing intermediates of viral satellite RNA replication in plants. A minimal hairpin ribozyme consists of two helix-loop-helix segments. These segments associate noncoaxially in the active folded structure in a way that brings catalytically important loop nucleotides into close proximity. The hairpin ribozyme in the satellite RNA of Tobacco Ringspot Virus assembles in the context of a four-way helical junction. Recent physical characterization of hairpin ribozyme structures using fluorescence resonance energy transfer demonstrated enhanced stability of the folded structure in the context of a four-way helical junction compared to minimal hairpin ribozyme variants. Analysis of the functional consequences of this modification of the helical junction has revealed two changes in the hairpin ribozyme kinetic mechanism. First, ribozymes with a four-way helical junction bind 3' cleavage products with much higher affinity than minimal hairpin ribozymes, evidence that tertiary interactions within the folded structure contribute to product binding energy. Second, the balance between ligation and cleavage shifts in favor of ligation. The enhanced ligation activity of hairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.

Binding Sites↗

PubMed1988

An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

Animals↗

PubMed1976

Surface immunoglobulins of lymphocytes in mouse plasmacytoma. IV. Evidence for the persistence of the effect of plasmacytoma-RNA on the surface immunoglobulins of normal lymphocytes in vivo and in vitro.

The previous findings were confirmed that RNA extracted from murine plasmacytoma alters the character of the lymphocyte surface immunoglobulins (SIg) to express the idiotypic specificity of the Ig of the plasmacytoma from which the RNA was derived (cell conversion). RNA extracted from spleens of plasmacytoma-RNA-injected BALB/c mice also had convering activity, and if injected into other mice, caused the appearance of RNA active in cell conversion in spleens of the second set of mice. This activity was lost only after two additional transfers. When splenic cells from animals 1 hr after injection with RNA extracted from MOPC 300, LPC-1 or MOPC 104E, were cultured for 7 days, the proportion of cells with the SIg specific for these tumours increased. The cell-converting activity of the RNA extracted from the cultured cells after 7 days incubation ('7-day' RNA) was higher than that of RNA extracted from cells after 1 hr incubation ('immediate' RNA). 'Seven-day' RNA could be used for sequential transfers without marked loss of activity in cell conversion for at least five transfers. The repetitive transferability of this phenomenon by the injection of plasmacytoma-RNA suggests the possibility of RNA replication in the recipient cells.

Animals↗

PubMed1979

Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down.

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.

Coliphages↗

PubMed2006

Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.

Adaptation, Biological↗

PubMed2001

Sequences at the 3' untranslated region of bamboo mosaic potexvirus RNA interact with the viral RNA-dependent RNA polymerase.

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555-565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain (Delta 893) derived from BaMV open reading frame 1 could specifically bind to the 3' UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3' UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3' UTR showed that Delta 893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Delta 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.

3' Untranslated Regions↗

PubMed1997

Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase.

Poly(rC) binding protein 2 (PCBP2) forms a specific ribonucleoprotein (RNP) complex with the 5'-terminal sequences of poliovirus genomic RNA, as determined by electrophoretic mobility shift assay. Mutational analysis showed that binding requires the wild-type nucleotide sequence at positions 20-25. This sequence is predicted to localize to a specific stem-loop within a cloverleaf-like secondary structure element at the 5'-terminus of the viral RNA. Addition of purified poliovirus 3CD to the PCBP2/RNA binding reaction results in the formation of a ternary complex, whose electrophoretic mobility is further retarded. These properties are consistent with those described for the unidentified cellular protein in the RNP complex described by Andino et al. (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598). Dicistronic RNAs containing mutations in the 5' cloverleaf-like structure of poliovirus that abate PCBP2 binding show a decrease in RNA replication and translation of gene products directed by the poliovirus 5' noncoding region in vitro, suggesting that the interaction of PCBP2 with these sequences performs a dual role in the virus life cycle by facilitating both viral protein synthesis and initiation of viral RNA synthesis.

3C Viral Proteases↗

PubMed2006

Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.

Animals↗

PubMed1992

LRV1 viral particles in Leishmania guyanensis contain double-stranded or single-stranded RNA.

The 32-nm-diameter spherical viral particles found in the cytoplasm of Leishmania guyanensis CUMC1-1A sediment at 130S and have a buoyant density of approximately 1.4 g/ml in cesium chloride gradients. These particles contain a 5.3-kb double-stranded RNA, while single-stranded RNA that corresponds to the viral positive strand is associated with less-dense particles. These results suggest a conservative and sequential mode of LRV1 viral RNA replication that is exemplified by the ScV L-A virus of yeast.

Animals↗

PubMed2000

Structure and function of the hairpin ribozyme.

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.

Animals↗

PubMed2001

Selection for fitness versus selection for robustness in RNA secondary structure folding.

I investigate the competition between two quasispecies residing on two disparate neutral networks. Under the assumption that the two neutral networks have different topologies and fitness levels, it is the mutation rate that determines which quasispecies will eventually be driven to extinction. For small mutation rates, I find that the quasispecies residing on the neutral network with the lower replication rate will disappear. For higher mutation rates, however, the faster replicating sequences may be outcompeted by the slower replicating ones if the connection density on the second neutral network is sufficiently high. The analytical results are in excellent agreement with flow-reactor simulations of replicating RNA sequences.

Base Sequence↗

PubMed1993

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.

Amino Acid Sequence↗