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At least 649 records · Page 36Linked to original sources
PubMed1992

Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.

Amino Acid Sequence↗
PubMed1999

H4 acetylation, XIST RNA and replication timing are coincident and define x;autosome boundaries in two abnormal X chromosomes.

The inactive X (Xi) differs from its active homologue (Xa) in a number of ways, including increased methylation of CpG islands, replication late in S phase, underacetylation of histone H4 and association with XIST RNA. Global changes in DNA methylation occur relatively late in development, but the other properties all change during or shortly after the establishment of Xi and may play a role in the mechanism by which an inactive chromatin conformation spreads across most of the chromosome. In the present report, we use two human X;autosome translocation chromosomes to study the spreading of inactive X chromatin across X;autosome boundaries. In one of these chromosomes, t(X;6), Xp distal to p11.2 is replaced by 6p21.1-6pter and, in the other, ins(X;16), a small fragment derived from 16p13 is inserted into the distal third of Xq. In lymphoid cells from patients carrying these translocations in an unbalanced form, Xi was shown by HUMARA assay to be derived exclusively [t(X:6)] or predominantly [ins (X;16)] from the derived X chromosome. We used a combination of immunolabelling and RNA/DNA fluorescence in situ hybridization to define the distribution of XIST RNA, deacetylated H4 and late-replicating DNA across the two derived X chromosomes in inactive form. Within the limits of the cytogenetic techniques employed, the results show complete coincidence of these three parameters, with all three being excluded from the autosomal component of the derived X chromosome.

Acetylation↗
PubMed2000

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Base Sequence↗
PubMed1994

Inhibition of vesicular stomatitis virus RNA synthesis by protein hyperphosphorylation.

Vesicular stomatitis virus (VSV) RNA synthesis requires the template nucleocapsid, the polymerase (L) protein, and the cofactor phosphorylated (P/NS) protein. To determine whether the degree of phosphorylation regulated VSV RNA synthesis, infected Chinese hamster ovary cells were treated with okadaic acid (OKA), a serine/threonine phosphatase inhibitor. OKA reduced viral penetration and uncoating but had little or no effect on primary transcription or viral protein synthesis. However, approximately 80% of total viral RNA synthesis was inhibited when 2 microM or more OKA was added to infected cells after viral uncoating had taken place. Analysis of proteins and RNA species in infected cells labeled with 32P showed that OKA led to hyperphosphorylation of two viral phosphoproteins, the P/NS protein and matrix protein (M), resulting in inhibition of full-length RNA synthesis and subsequent secondary transcription. Pulse-chase experiments demonstrated that the hyperphosphorylated P/NS species was converted rapidly from the less phosphorylated form. Hyperphosphorylated P/NS as well as the less phosphorylated form, but not M, were found to be associated with nucleocapsids isolated from cytoplasmic extracts. These results suggest that phosphorylation played an important role in the regulation between viral transcription and viral RNA replication as well as the turning off of RNA replication. Thus, phosphatase inhibitors promise to be a valuable tool for dissecting the regulatory mechanisms involving phosphorylated viral proteins.

Animals↗
PubMed1975

Nucleotide sequence of microvariant RNA: another small replicating molecule.

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.

Base Sequence↗
PubMed1987

Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins.

Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.

Antibody Specificity↗
PubMed2004

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5.

BACKGROUND: Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated. RESULTS: To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication. CONCLUSIONS: The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.

Gene Expression Regulation, Viral↗
PubMed1983

A host protein (La) binds to a unique species of minus-sense leader RNA during replication of vesicular stomatitis virus.

Baby hamster kidney cells infected with the minus-strand RNA virus vesicular stomatitis virus (VSV) were found to contain three small viral leader RNA species of the minus sense. The longest minus-strand leader RNA was 54 nucleotides long and was complexed with the host cell La protein that was immunoprecipitated by antisera from patients with systemic lupus erythematosus. The La protein is normally found associated with RNA polymerase III transcripts in their unprocessed form. Shorter minus-strand leader RNA species of 45-48 nucleotides were more abundant but were not associated with the La protein. Unlike the plus-strand leader RNA of VSV, the minus-strand leader RNAs were not detected in the nucleus in any form. The minus-strand leader RNAs accumulated gradually throughout the infection and could not be found in association with the viral nucleocapsid protein. The sequence required for La protein binding on the 54-nucleotide-long minus-strand leader is similar to that at the 3' end of the La protein binding-plus-strand leader RNA and, thus, we propose a role for the La protein in the replication of VSV.

Animals↗
PubMed2001

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants.

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

Base Sequence↗
PubMed1988

Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase.

In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs. In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA. This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function. Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein. The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase. By employing an E. coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred. This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.

Base Sequence↗
PubMed2001

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Base Sequence↗
PubMed2006

Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication.

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Amino Acid Sequence↗
PubMed2000

Requirement of a 5'-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with turnip crinkle virus.

Viral RNA replication begins with specific recognition of cis-acting RNA elements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated host factors. A short RNA element (3'-AACCCCUGGGAGGC) located 41 bases from the 5' end of minus strands of satellite RNA C (satC), a 356-base subviral RNA naturally associated with turnip crinkle virus (TCV), was previously identified as important for plus-strand synthesis using an in vitro RdRp assay (H. Guan, C. Song, A. E. Simon, 1997, RNA 3, 1401-1412). To examine the functional significance of this element in RNA replication, mutations were introduced into the consecutive C residues in the element. A single mutation of the 3'-most C residue resulted in undetectable levels of satC plus strands when transcripts were assayed in protoplasts and suppressed transcription directed by the element in vitro. However, satC minus strands were detectable at 6 h postinoculation (hpi) of protoplasts, accumulating to about 10% of wild-type levels at 24 hpi. This mutation, when in the plus-sense orientation, had little or no effect on minus-strand synthesis from full-length satC plus strands in vitro, suggesting that the 5'-proximal RNA element is required for satC plus-strand synthesis. In addition, in vivo genetic selection revealed a strict requirement for 10 of the 14 nucleotides of the element, indicating that the primary sequence is essential for RNA accumulation.

5' Untranslated Regions↗
PubMed1983

Replication of vesicular stomatitis virus defective interfering particle RNA in vitro: transition from synthesis of defective interfering leader RNA to synthesis of full-length defective interfering RNA.

The replication of the RNA of vesicular stomatitis virus (VSV) defective interfering (DI) particles was established in a defined cell-free system. The transition from synthesis of only the DI-leader RNA to replication of the full-length DI RNA was effected in the system by newly synthesized VSV proteins and occurred in the absence of VSV helper virus. Both positive- and negative-polarity full-length DI RNA were synthesized. Furthermore, the products of RNA replication associated with newly synthesized viral proteins to form complexes that were indistinguishable from authentic DI particle nucleocapsids on the basis of buoyant density and resistance to ribonuclease digestion. The DI-leader RNA did not form ribonuclease-resistant structures. We conclude that this in vitro system successfully executes many of the reactions of VSV DI particle replication and assembly.

Capsid↗
PubMed2005

Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

Increasing evidence indicates that RNA interference (RNAi) may be used to provide antiviral immunity in mammalian cells. Human micro (mi)RNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN)-induced protein kinase R (PKR) but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si) and micro (mi)RNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

HIV↗
PubMed2001

Specific inhibition of coxsackievirus B3 translation and replication by phosphorothioate antisense oligodeoxynucleotides.

The 5' and 3' untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary structures that have been confirmed to play important regulatory roles in viral cap-independent internal translation initiation and RNA replication. We previously demonstrated that deletions in different regions of the 5' UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides (AS-ODNs) were applied to target crucial sites within the 5' and 3' UTRs. In this study, seven phosphorothioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR, and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3 were strongly inhibited by the AS-ODNs complementary to different regions of the 5' and 3' UTRs. The most effective sites are located at the proximate terminus of the 5' UTR (AS-1), the proximate terminus of the 3' UTR (AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region (AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with AS-1 and AS-7 targeting the termini of the 5' and 3' UTRs. The percent inhibition values of AS-1 and AS-7 for CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7, respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.

Enterovirus B, Human↗
PubMed1997

Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication.

The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.

Animals↗