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Results for “RNA Replication”

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PubMed2004

Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Aminoglycosides↗

PubMed1992

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR.

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Animals↗

PubMed1964

REPLICATION OF VIRAL RNA, III. DOUBLE-STRANDED REPLICATIVE FORM OF MSW PHAGE RNA.

Explore the source record for details and available documents.

Adenosine Triphosphate↗

PubMed1988

Inhibitory effect of sulphur-containing purine nucleoside analogues on replication of RNA viruses: selective antiviral activity against influenza viruses.

Four sulphur-containing purine nucleoside analogues: 6MP, 6-thioinosine, 6-methylthioinosine and 6-ethylthioinosine, were examined for antiviral activity against some RNA viruses. All compounds extensively inhibited the replication of influenza viruses but had no inhibitory effect on other RNA viruses: Sendai, RS, vesicular stomatitis and western equine encephalitis viruses.

Amidophosphoribosyltransferase↗

PubMed1993

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.

Animals↗

PubMed1992

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.

Animals↗

PubMed1996

Fluctuations in steady state level of genomic HIV-1 RNA and replication intermediates related to disease progression rate.

Steady state levels of HIV-1 genomic RNA and unintegrated circular DNA fluctuate in the course of HIV-1 infection. Genomic RNA in serum was detected using the NASBA technique and the amount of circular DNA was assessed by PCR. Quantification was done by competitive techniques using co-amplification of internal standards. Within the Amsterdam Cohort Studies it was possible to distinguish rapid progressors, intermediate progressors, slow progressors and non-progressors. Rapid progressors show persistently high viral RNA loads from seroconversion on, while all other HIV-1-infected individuals show a steady decline after seroconversion. Subsequent rises in viral RNA levels herald disease progression in later stages of infection. Unintegrated circular DNA shows similar, but somewhat delayed kinetics. These results indicate that the distribution of AIDS and the average length of the symptom-free period in an HIV-1-infected host population is determined by the steady state levels of genomic RNA and of replication intermediates that are produced by a particular HIV-1 virus population in the average seropositive individual.

Biomarkers↗

PubMed2003

Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication.

The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.

Blotting, Northern↗

PubMed2003

Analysis of the cloverleaf element in a human rhinovirus type 14/poliovirus chimera: correlation of subdomain D structure, ternary protein complex formation and virus replication.

RNA genomes of enteroviruses and rhinoviruses contain a 5'-terminal structure, the cloverleaf (CL), which serves as signal in RNA synthesis. Substitution of the poliovirus [PV1(M)] CL with that of human rhinovirus type 2 (HRV2) was shown previously to produce a viable chimeric PV, whereas substitution with the HRV14 CL produced a null phenotype. Fittingly, the HRV14 CL failed to form a complex with PV-specific proteins 3CD(pro)-3AB or 3CD(pro)-PCBP2, considered essential for RNA synthesis. It was reported previously (Rohll et al., J Virol 68, 4384-4391, 1994) that the major determinant for the null phenotype of a PV/HRV14 chimera resides in subdomain Id of the HRV14 CL. Using a chimeric PV/HRV14 CL in the context of the PV genome, stem-loop Id of HRV14 CL was genetically dissected. It contains the sequence C(57)UAU(60)-G, the underlined nucleotides forming the loop that is shorter by 1 nt when compared to the corresponding PV structure (UUGC(60)GG). Insertion of a G nucleotide to form a tetra loop (C(57)UAU(60)GG(61)) did not rescue replication of the chimera. However, an additional mutation at position 60 (C(57)UAC(60)GG(61)) yielded a replicating genome. Only the mutant PV/HRV14 CL with the UAC(60)G tetra loop formed ternary complexes efficiently with either PV proteins 3CD(pro)-3AB or 3CD(pro)-PCBP2. Thus, in the context of PV RNA synthesis, the presence of a tetra loop in subdomain D of the CL per se is not sufficient for function. The sequence and, consequently, the structure of the tetra loop plays an essential role. Biochemical assays demonstrated that the function of the CL element and the function of the cis-acting replication element in the 3D(pol)-3CD(pro)-dependent uridylylation of VPg are not linked.

Base Sequence↗

PubMed2003

Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins.

Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS-PAGE and MALDI-TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.

Amino Acid Sequence↗

PubMed2003

Differential inhibition of RNA editing in hepatitis delta virus genotype III by the short and long forms of hepatitis delta antigen.

Hepatitis delta virus (HDV) produces two essential forms of the sole viral protein from the same open reading frame by using host RNA editing activity at the amber/W site in the antigenomic RNA. The roles of these two forms, HDAg-S and HDAg-L, are opposed. HDAg-S is required for viral RNA replication, whereas HDAg-L, which is produced as a result of editing, inhibits viral RNA replication and is required for virion packaging. Both the rate and amount of editing are important because excessive editing will inhibit viral RNA replication, whereas insufficient editing will reduce virus secretion. Here we show that for HDV genotype III, which is associated with severe HDV disease, HDAg-L strongly inhibits editing of a nonreplicating genotype III reporter RNA, while HDAg-S inhibits only when expressed at much higher levels. The different inhibitory efficiencies are due to RNA structural elements located ca. 25 bp 3' of the editing site in the double-hairpin RNA structure required for editing at the amber/W site in HDV genotype III RNA. These results are consistent with regulation of amber/W editing in HDV genotype III by a negative-feedback mechanism due to differential interactions between structural elements in the HDV genotype III RNA and the two forms of HDAg.

Cell Line↗

PubMed1988

Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus.

Complete cDNA copies of genomic RNA1, RNA2, and RNA3 of cowpea chlorotic mottle virus (CCMV) were cloned 1 base downstream from a T7 RNA polymerase promoter. The mixture of capped in vitro transcripts from all three clones produced normal CCMV infections in barley protoplasts and cowpea plants. By using transcripts from these clones and from a similar set of biologically active clones of the related brome mosaic virus (BMV), all possible single component exchanges between the BMV and CCMV tripartite genomes were tested. Viral RNA replication was not observed with any heterologous combination of RNA1 and RNA2, which encode trans-acting viral RNA replication factors. However, substitution of the heterologous RNA3 into either genome produced viable hybrid viruses, both of which replicated in barley protoplasts and produced lesions on Chenopodium hybridum, a local lesion host for both parent viruses. In hybrid infections, BMV and CCMV coat proteins each readily packaged RNAs from the heterologous virus, but BMV RNAs were replicated to a higher level than CCMV RNAs, even when trans-acting RNA replication factors were provided by CCMV genes. Neither hybrid systemically infected the natural host of either parent virus, suggesting that host specificity determinants in BMV and CCMV are encoded by RNA3 and at least one other genomic RNA.

Base Sequence↗

PubMed1991

Interference in trans with brome mosaic virus replication by RNA-2 bearing aminoacylation-deficient mutants.

The tRNA-like domain present at the 3' end of each of the three genomic RNAs of brome mosaic virus (BMV) encompasses the (-)-strand promoter essential for replication. The replicative competence of two BMV RNA-2 transcripts bearing mutations delta 5' and 5'AGA in the tRNA-like domain (previously shown by in vitro assays to be deficient in tyrosylation) was evaluated in barley protoplasts. Transfection of protoplasts with low (2 micrograms) amounts of delta 5'RNA-2, together with transcripts of wild-type RNA-1 and -3, not only incapacitated the replication of RNA-2 but also significantly interfered in trans with the synthesis and accumulation of the other viral RNAs. In contrast, RNA-2 mutants bearing either 5'AGA or M4 (a mutation yielding enhanced minus-strand replication activity in vitro) were inhibitory to viral replication only when present at a relatively high level (12 micrograms). Coinoculation of protoplasts with high levels (12 micrograms) of each of the three RNA-2 mutants and transcripts corresponding to wild-type RNA-1, -2 and -3 (2 micrograms each) revealed that the mutants were capable of competing in trans, resulting in greatly reduced accumulation of the viral RNA and suggesting that their expression from constitutive promoters in transgenic plants may provide protection against viral infection.

Base Sequence↗

PubMed1997

A specific host cellular protein binding element near the 3' end of mouse hepatitis virus genomic RNA.

A distinct host cellular protein binding element was mapped within a 38-nucleotide (nt) sequence 166-129 nucleotides upstream of the 3' end of the MHV-JHM genome using a RNase T1 protection/gel mobility shift electrophoresis assay. The resultant RNA-protein complex contains six host cellular proteins, one protein of 120-kDa molecular mass, two poorly resolved species approximately 55 kDa in size, a second pair of poorly resolved 40-kDa proteins, and a minor component of 25 kDa. A series of RNA probes containing deletions or clustered transversion mutations were tested for their ability to form complexes with mock- and MHV-JHM-infected cytoplasmic extracts. Three mutant RNA probes (mA, mB, and mC) with deletions at 154-140, 139-129, and 128-118, respectively, expressed 4, 37, and 94% of the host protein binding activity exhibited by the wild-type RNA. Defective interfering (DI) RNAs (DImA, DImB, and DImC) containing corresponding deletions at 154-140, 139-129, 128-118, and another DI RNA (DImD) with a deletion at nucleotides (nts) 112-102, a region which did not affect RNA-protein interactions, were transfected into MHV-JHM-infected 17CL-1 cells to assay the effects of these mutations on DI RNA replication. All of these mutations had an adverse effect on DI RNA replication. However, analysis of negative strand mutant DI RNAs revealed that two mutants (DImC and DImD) carrying deletions having little or no effect on RNA-protein interaction in our RNA-protein binding assays maintained their mutant sequences. In contrast, the other two mutants (DImA and DImB) containing deletions that dramatically decreased RNA-protein binding activity did not maintain their mutations; wild-type sequences were restored in the majority of the progeny negative strand molecules. These data indicate that the 26-nucleotide sequence at positions 154-129 from the 3' end of viral genome is important to both RNA-protein binding and viral replication. This protein binding element contains an 11-nt sequence (UGAGAGAAGUU, positions 139-129) very similar to a more 3' sequence (UGAAUGAAGUU) previously implicated in host protein binding and viral RNA replication (Yu and Leibowitz, 1995a and 1995b).

Animals↗

PubMed2000

Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis.

Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.

Coated Vesicles↗

PubMed2000

The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 10(45)-fold amplification.

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85-95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura(+) colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

Bromovirus↗

PubMed2001

Secondary structural elements within the 3' untranslated region of mouse hepatitis virus strain JHM genomic RNA.

Previously, we characterized two host protein binding elements located within the 3'-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3'-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).

3' Untranslated Regions↗

PubMed2002

DNA replication and RNA synthesis in thymocyte nuclei microinjected into the cytoplasm of artificially activated mouse eggs.

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

Animals↗