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At least 937 records · Page 52Linked to original sources
PubMed1975

Virus-specific proteins associated with the replication complex of poliovirus RNA.

The poliovirus replication complex was isolated and purified from infected HeLa S3 cells. Preparations with RNA-dependent RNA polymerase activity were concentrated 200- and 1000-fold with respect to the original virus and total protein content. The enzyme activity was found to be associated with the proteins NCVPI, 2, 3, 4, (5), 6 and VPl/NCVPx. The structural proteins VP2, 3 and 4 were not present. Addition of cycloheximide to infected cells resulted in a decrease in the in vitro polymerase activity and a loss in NCVPI content. Treatment of the infected cells with toloylsulphonyl-phenylalanine chloromethyl ketone (TPCK) and iodoacetamide (IAA) led to an inhibition of in vivo RNA synthesis. The 750 g supernatant fluids obtained from extracts of these cells were able to block RNA synthesis in vitro. Electrophoretic profiles of the respective protein compositions indicate that large virus precursor proteins are responsible for the inhibition of poliovirus RNA synthesis in vivo and in vitro.

Cycloheximide↗
PubMed1995

Design of artificial short-chained RNA species that are replicated by Q beta replicase.

Different RNA species that are replicated by Q beta replicase have related secondary structures: for both plus and minus strands, "leader" stem structures were found at their 5' termini, while their 3' termini were unpaired. Parallel structures in complementary strands rather than antiparallel ones require the occurrence of wobble pairs and other imperfections in the stem regions. To test whether the leader structures are required for replication, artificial RNA sequences were synthesized by transcription from synthetic oligodeoxynucleotides with T7 RNA polymerase and assayed for their ability to be replicated by Q beta replicase. A synthetic short RNA species known to be replicated was amplified, forming a stable quasi-species; i.e., its sequence was conserved during hundreds of replication rounds. A synthetic mutant of this sequence that stabilized the leader in one strand but favored a 3'-terminal stem in the other one led to the complete loss of template activity. When new RNA sequences with the described structural requirements were designed and synthesized, their template activity was too low to be directly measurable; however, incubation with replicase produced replicating RNA whose sequence was closely related to the synthesized RNA species. The most likely interpretation is that the designed sequences were in a low montainous region in the replication fitness landscape and were optimized during amplification by Q beta replicase to a nearby fitness peak. The structural features postulated to be required for replication were not only conserved but even improved in the outgrowing mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2006

Restoration in vivo of defective hepatitis delta virus RNA genomes.

The 1679-nt single-stranded RNA genome of hepatitis delta virus (HDV) is circular in conformation. It is able to fold into an unbranched rodlike structure via intramolecular base-pairing. This RNA is replicated by host RNA polymerase II (Pol II). Such transcription is unique, because Pol II is known only for its ability to act on DNA templates. The present study addressed the ability of the HDV RNA replication to tolerate insertions of up to 1000 nt of non-HDV sequence either at an end of the rodlike RNA structure or at a site embedded within the rod. The insertions did not interfere with the ability of primary transcripts to be processed in vivo by ribozyme cleavage and ligation. The insertions greatly reduced the ability of genomes to replicate. However, when total RNA from such transfected cells was used to transfect new recipient cells, replicating HDV RNAs could be detected by Northern analyses. The size of the emerged RNAs was consistent with loss of the inserted sequences. RT-PCR, cloning, and sequencing showed that recovery involved removal of inserted sequences with or without small deletions of adjacent RNA sequences. Such restoration of the RNA genome is consistent with a model requiring intramolecular template-switching (RNA recombination) during RNA-directed transcription, in combination with biological selection for maintenance of the rodlike structure of the template.

Base Pairing↗
PubMed2002

Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees.

The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo.

Adaptation, Physiological↗
PubMed1984

Sites of copy choice replication involved in generation of vesicular stomatitis virus defective-interfering particle RNAs.

The copy choice model for the generation of defective interfering (DI) particles of vesicular stomatitis virus suggests that during replication the polymerase prematurely terminates, moves with the nascent daughter strand to another site on the same or a different template molecule, and resumes elongation of the nascent chain. We have analyzed the sites where premature termination or resumption of replication has occurred during the generation of the deletion DI particle LT, the snapback DI particle 011, and the panhandle DI particles T, T(L), and 611. The recombination sites were identified by comparing the nucleotide sequences of the relevant regions of these DI particle RNAs to those of the vesicular stomatitis virus L gene (Schubert et al., J. Virol. 51:505-514, 1984). Sequence homology was not detected between these sites, which rules out the existence of a general terminator or promoter sequence involved in copy choice replication. In several cases, however, premature termination or resumption of RNA replication may be favored by specific signal sequences. The sequences immediately before the start and at the end of the deletion in DI LT contain two hexanucleotides, ATCTGA and GATTGG, in a similar spacing. In these case of DI T and 611, but not of DI T(L), the end of the 5'-terminal region bears the hexanucleotide CCUCUU. This sequence is also repeated in the stem region in all three DI particle genomes. In addition, we present data that the added 3'-terminal regions of the panhandle DI particle RNAs may differ by only one base and are 46 [DI T(L) and 611] or 45 (DI T) bases long. We suggest that each site of the vesicular stomatitis virus genome has the potential to give rise to DI particle RNAs. Specific sequences, however, may modulate this process in a quantitative way, and they favor the generation of certain types of DI particle genomes like those of the panhandle type.

Base Sequence↗
PubMed1997

Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.

Base Sequence↗
PubMed2005

Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B.

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.

Amino Acid Sequence↗
PubMed1988

Replicase of L-A virus-like particles of Saccharomyces cerevisiae. In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form.

Virus-like particles that contain L-A double-stranded RNA are known to have transcriptase activity whose product is L-A single-stranded plus RNA. In low salt conditions, these particles release their double-stranded RNA and can then use added plus L-A or plus M1 single-stranded RNAs as templates to synthesize their respective double-stranded RNAs. The reaction requires dialyzed L-A virus-like particles as the source of the enzyme, a partially purified cell extract (host factor(s)), added single-stranded RNA as a template, and polyethylene glycol 6000, along with four NTPs. Crude host factor extracts prepared from mak3 or mak10ta mutants also support the reaction as effectively as that from a wild type strain, while a crude extract prepared from a pet18 mutant grown under the nonpermissive conditions is less effective. Template specificity of the in vitro reaction is the same as that expected for the enzyme reaction in vivo. Plus L-A and plus M1 single-stranded RNAs, but not 18 S rRNA, are converted to their respective double-stranded RNAs with net RNA synthesis. The newly synthesized strand of M1 double-stranded RNA is a full-length minus strand. This demonstration of replicase activity in the mature L-A virus-like particles which contain L-A double-stranded RNA is consistent with our previous L-A double-stranded RNA replication model; the difference between the mature L-A virus-like particles and L-A double-stranded RNA-synthesizing particles (expected to be replication intermediates in vivo) is just that the former contain L-A double-stranded RNA, while the latter contain L-A plus single-stranded RNA.

DNA Replication↗
PubMed2006

Synthesis of minus-strand copies of a viral transgene during viral infections of transgenic plants.

Viral transgenes designed to provide resistance to specific plant viruses frequently consist of the coat protein gene and a contiguous 3' untranslated region (3'UTR) of viral origin. In many RNA viruses the viral 3'UTR establishes a recognition and initiation site for viral RNA replication. Thus the transgenic transcript may contain a functional virus replication site. Experiments were designed to determine if a challenging virus would recognize this replication site on a nuclear derived transcript and synthesize the complementary RNA. These data demonstrate that upon infection by a virus that recognizes the viral replication site, a full-length complement of the transgenic transcript is produced. In these experiments the replication complex of Brome Mosaic bromovirus recognized the transgenic transcript derived from a Cowpea Chlorotic Mottle bromovirus transgene. The resulting RNA may contribute to RNA recombination events.

Bromovirus↗
PubMed2004

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core.

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

Acid Anhydride Hydrolases↗
PubMed1984

Studies on the mechanism of RNA synthesis of a murine coronavirus.

The mechanism of viral RNA replication in mouse hepatitis virus (MHV)-infected cells was studied by oligonucleotide mapping of every mRNA. We discovered that an oligonucleotide, No. 10, was localized at the 5'-end of every mRNA, and was not colinear with the sequences of the virion genomic RNA. This result indicates that all of the mRNAs contain a leader sequence which is joined to the body sequences of the mRNAs. We have also studied the structure of the replicative intermediate (RI) RNA in the MHV-infected cells. This RI RNA consists of a single species corresponding to the MHV genomic RNA. No subgenomic RI structures were detected. Furthermore, the nascent RNA chains in the RI structure contained the leader sequences, suggesting that the leader RNA was not added to the mRNA post- transcriptionally , but rather, it was probably synthesized independently and then used as a primer for the synthesis of mRNAs. We have also shown that the poly (A) sequences in the MHV genome were transcribed from the poly (U) sequences present in the negative-strand template. The RNA polymerases involved in the MHV RNA synthesis were also characterized. The early polymerase synthesizes a single negative-stranded, full-length RNA. The late polymerases could be separated into two activities, one synthesizing positive-stranded genomic RNA, and the other synthesizing genomic as well as subgenomic RNAs. Thus, the replication and transcription functions of MHV could probably be separated. A plausible model of MHV replication is presented.

Animals↗
PubMed1987

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.

Animals↗
PubMed1976

A genetic approach to studies on the synthesis of the complementary RNA's of fowl plague virus.

The rate of incorporation of 5-fluorouracil into complementary strands of replicating RNA of fowl plague virus (FPV) has been studied. The efficiency of incorporation was estimated by determination of the reversion frequency in s-mutants with known types of base transitions in the RNA of the virus particle. It was established that maximum incorporation of 5-fluorouracil into progeny virus particle RNA took place between 2 and 4 h after infection. The maximum incorporation of the mutagen into complementary RNA (plus strands) occurred when the cells were exposed to 5-fluorouracil from 1 to 2 h after infection.

Base Sequence↗
PubMed1998

Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features.

BACKGROUND: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an 'RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. RESULTS: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular ('trans') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. CONCLUSIONS: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the 'helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.

Adenosine Monophosphate↗
PubMed2006

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection.

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.

Animals↗
PubMed2000

Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus.

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

Bromovirus↗
PubMed2005

Pokeweed antiviral protein inhibits brome mosaic virus replication in plant cells.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

Base Sequence↗