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At least 667 records · Page 37Linked to original sources
PubMed1993

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.

Animals↗
PubMed2003

Replication complex of human parechovirus 1.

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

Amino Acid Sequence↗
PubMed1996

Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.

DNA, Complementary↗
PubMed2002

Modulation of HIV-1 replication by RNA interference.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Cell Line↗
PubMed1988

Structure and protein composition of the rotavirus replicase particle.

To understand the role of viral proteins in the replication of rotavirus RNA, we have characterized the structure of subviral particles (SVPs) that synthesize double-stranded RNA (DS RNA). Pulse-labeling of newly made RNA in infected cells showed that rotavirus DS RNA was synthesized either in single-shell (SS)-like particles or in precursor particles that rapidly mature into SS particles. Experiments using a cell-free system demonstrated that most replicase particles containing newly made DS RNA were of greater density in CsCl than single-shelled (SS) particles. However, this was partly due to the presence of single-stranded RNAs as the treatment of replicase particles with micrococcal nuclease reduced their density to between core particles and SS particles. Electrophoretic analysis indicated that replicase particles, purified by centrifugation on CsCl and glycerol gradients, were similar to SS particles, containing the structural proteins VP1, VP2, and VP6. Rotavirus replicase particles were also found to contain the nonstructural proteins NS34 and NS35 and possible host components. The presence of VP6 in enzymatically active replicase particles suggests that, like transcription, this protein may be required for rotavirus RNA replication.

Capsid↗
PubMed1990

Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribed in sense and antisense orientation from the 5'-leader/gag region.

The inhibitory effects of expression plasmids on HIV-1 replication were studied in a transient assay system. Test plasmids were co-microinjected with non-defective proviral HIV-1 DNA into a colon-carcinoma cell line (SW480) and the resulting infectious HIV-1 was quantitated after amplification in cocultivated CD4+ MT-4 cells. At a molar ratio of 1:1 and 5:1 plasmids capable of expressing a 410 bp HIV-1 fragment as antisense or sense transcript respectively both specifically inhibited HIV-1 replication up to 70%. This effect was specific for HIV-1 sequences and was not observed upon expression of unrelated RNA-segments. At a molar excess equal to or greater than 15:1, additional inhibitory effects were seen with control plasmids carrying only the strong human cytomegalovirus immediate early (HCMV IE) promoter/enhancer element. The reasons for these findings are discussed.

CD4 Antigens↗
PubMed2001

The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.

Animals↗
PubMed1988

Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.

Animals↗
PubMed2004

Integrative analysis of genome-scale data by using pseudoinverse projection predicts novel correlation between DNA replication and RNA transcription.

We describe an integrative data-driven mathematical framework that formulates any number of genome-scale molecular biological data sets in terms of one chosen set of data samples, or of profiles extracted mathematically from data samples, designated the "basis" set. By using pseudoinverse projection, the molecular biological profiles of the data samples are least-squares-approximated as superpositions of the basis profiles. Reconstruction of the data in the basis simulates experimental observation of only the cellular states manifest in the data that correspond to those of the basis. Classification of the data samples according to their reconstruction in the basis, rather than their overall measured profiles, maps the cellular states of the data onto those of the basis and gives a global picture of the correlations and possibly also causal coordination of these two sets of states. We illustrate this framework with an integration of yeast genome-scale proteins' DNA-binding data with cell cycle mRNA expression time course data. Novel correlation between DNA replication initiation and RNA transcription during the yeast cell cycle, which might be due to a previously unknown mechanism of regulation, is predicted.

Cell Cycle↗
PubMed1981

Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing.

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region (P3) of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occurs between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.

Amino Acid Sequence↗
PubMed2002

The synthesis of minus-strand RNA of bamboo mosaic potexvirus initiates from multiple sites within the poly(A) tail.

The 3' terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5' portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3' untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5' terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5' rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5'-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5' RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3' UTR of BaMV genomic RNA.

5' Untranslated Regions↗
PubMed2005

Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.

Amino Acid Sequence↗
PubMed1996

Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway.

The entry pathway of porcine reproductive and respiratory syndrome virus (PRRSV) into MARC-145 cells was investigated using a variety of drugs that interfere with the pH of intracellular vesicles by different mechanisms. Virus entry was assessed by measuring viral RNA replication or production of infectious virus. Chloroquine, ammonium chloride and bafilomycin A1 inhibited RNA replication or production of infectious virus in a dose-dependent manner. These drugs inhibited virus replication when added to the cells prior to, at infection or soon after infection. Moreover, the effect of chloroquine on PRRSV replication was reversible under acidic conditions of the media. Taken together, these results indicated that a low pH was required during virus entry. Electron microscopic data showed virus particles at the cell surface or within small vesicles which were circumscribed by a clathrin-like zone. In addition, the number of PRRSV-infected cells was decreased in the presence of cytochalasin B and phenylarsine oxide. Thus, we concluded that PRRSV entry might occur through a microfilament-dependent endocytic mechanism in which a low pH is necessary for proper virus uncoating.

Actin Cytoskeleton↗