Synthesis in vitro of RNA complementary to parental viral RNA by RNA polymerase induced by influenza virus.
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Adenosine Triphosphate↗
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Results for “RNA, VIRAL”
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Newcastle disease virus RNA. II. Preferential synthesis of RNA complementary to parental viral RNA by chick embryo cells.
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Adenine↗
Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: comparison with other viral and cellular RNA species.
Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 x 10(6) to 3.4 x 10(6) daltons and that of class b into the range of 2.2 x 10(6) to 2.9 x 10(6) daltons, depending on the standards used. The possible biological significance of this difference is discussed.
Alpharetrovirus↗
Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis.
Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.
Animals↗
Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.
The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.
Base Sequence↗
Polyadenylation and reverse transcription of influenza viral RNA.
The polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E. coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported. We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase. The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA. Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels. These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end. Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8.
DNA, Viral↗
Cell-to-cell transmission of human immunodeficiency virus infection induces two distinct phases of viral RNA expression under separate regulatory control.
A cell clone persistently infected with human T cell-lymphotrophic virus type IIIB (H3B cells) contained mainly the multiply spliced (2 kb) and singly spliced (4.3 kb) species of human immunodeficiency virus (HIV) RNA. When H3B cells were co-cultured with susceptible HUT78 cells, cell fusion occurred within 4 h of cell mixing and was accompanied by a marked increase of the unspliced full-length (9.2 kb) HIV RNA. This first phase of viral RNA induction (4 to 12 h post-infection) was followed by a second phase of viral RNA synthesis from 24 h p.i. in which there were significant increases in all three species of HIV RNA. Reverse transcriptase (RT) inhibitors such as azidothymidine (AZT) at concentrations that abolished de novo HIV DNA synthesis, abolished the first phase but not the second phase of viral RNA synthesis in our model system. A comparable one-step cell-free virus infection showed a pattern of viral RNA synthesis similar to that of the cell-to-cell transmission of infection. However, viral RNA synthesis following cell-free virus infection was totally inhibited by RT inhibitors. The early phase (4 to 12 h) expression of 9.2 kb HIV RNA is likely to use newly synthesized HIV DNA as template; during this phase, HIV RNA and DNA syntheses occur simultaneously, with each process being dependent on the other for maximal yield. During the later (24 to 48 h) phase, all three HIV RNA species may be transcribed at least in part from proviral DNA from the original donor cells. This later phase may provide one of the mechanisms for natural spread of virus to new cells and for enhanced viral gene expression in vivo, despite the presence of AZT.
Cell Line↗
Encephalomyocarditis virus replication complexes that prefer nucleoside diphosphates as substrates for viral RNA synthesis.
Replication complexes (RC) containing endogenous viral RNA templates and the viral RNA polymerase were isolated from encephalomyocarditis (EMC) virus-infected Krebs II cells by two different procedures one of which included removal of the bulk of the associated proteins by treatment with 2 M LiCl. Replacement of one or all the four nucleoside triphosphate (NTP) substrates by corresponding nucleoside diphosphates (NDPs) did not eliminate the ability of the RC to synthesize viral RNA products. Moreover, the complexes were shown to accept as substrates even nucleoside monophosphates (NMPs), provided at least one NTP was present in the system. These results suggested that nucleotide kinases, NMP kinase and NDP kinase, were associated with the RC, and this suggestion was confirmed directly. The RC could be resolved, by sucrose gradient centrifugation, into distinct components that exhibited marked preference for either NDPs or NTPs as substrates for RNA synthesis. It is suggested that, in the NDP-preferring components, the NTP molecules generated by the built-in nucleotide kinase system are channeled directly to the vicinity of the replication fork.
Adenosine Diphosphate↗
Characterization of a foot-and-mouth disease mutant temperature-sensitive for viral RNA synthesis.
A temperature-sensitive (ts) mutant of foot-and-mouth disease virus (FMDV) did not produce RNA polymerase activity nor synthesize viral RNA when incubated in cells solely at the nonpermissive temperature (38.5 degrees C). Infected cells initially incubated at 38.5 degrees C and then shifted down to 33 degrees C synthesized increased amounts of viral RNA at earlier times compared to infected cells kept at 33 degrees C throughout, indicating that RNA polymerase precursors were synthesized at 38.5 degrees C. In cells shifted up to 38.5 degrees C from 33 degrees C, the total amount of viral RNA synthesized after infection increased sharply for about 15 minutes and then rapidly increased over the next 2 hours. RNA polymerase activity presented a similar pattern in its initial twofold increase and subsequent rapid decrease. Pulse labeling experiments showed that mutant viral RNA synthesis continued at a diminishing rate for 2 hours in cells shifted up to 38.5 degrees C. The data from temperature after shift-up was degraded. The FMDV ts mutant is apparently additionally defective in being unable to protect viral RNA synthesized after shift-up to 38.5 degrees C.
Aphthovirus↗
Infectious bursal disease viral RNA amplification using RT/PCR from bursa tissue following phenol: chloroform inactivation of the virus.
The preservation of viable infectious agents for future studies could create complicated logistic problems, and at times it is not feasible. Methods for preserving the genetic integrity of inactivated agents would not only facilitate these studies but would also make it possible to transport inactivated preparations around the world. In this report, the effect of inactivation on the genetic material of infectious bursal disease virus (IBDV) was studied. Tissues from the bursa of Fabricius of birds experimentally infected 3 days earlier with the classic STC strain of IBDV were collected and immediately placed in a solution of phenol:chloroform:isoamyl alcohol (25:24:1) for 24, 48, 72, or 96 hr. Infected bursal tissue not treated with the phenol:chloroform solution and uninfected phenol: chloroform bursal tissue were used as controls. In a separate experiment, bursal tissues collected 5 days following infection of specific-pathogen-free birds with the classic STC or variant 1084-E strain were placed in the phenol:chloroform solution for 2 wk. All bursal samples were tested for viable IBDV following phenol:chloroform treatment. The tissues were washed in phosphate-buffered saline to remove phenol and then homogenized. Viability of the viruses in homogenized bursal tissue was examined by inoculation of embryonated chicken eggs. Viable IBDV was not observed in any phenol:chloroform-treated bursal tissue but was observed in the infected but non-phenol:chloroform-treated control bursa. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to test the integrity of the viral RNA. Viral RNA from the nontreated control and all infected bursal samples treated with phenol:chloroform solution at all the time points examined were transcribed into DNA, and a 394-bp fragment of the VP2 gene was amplified using specific primers in the PCR. The RT/PCR assay was negative using the phenol:chloroform-treated uninfected bursal tissue. This study clearly demonstrated that phenol:chloroform treatment of infected bursal tissue inactivated the classic and variant IBDV strains tested and preserved the viral RNA for use in the RT/PCR assay.
Animals↗
Sodium butyrate treatment of cells latently infected with HIV-1 results in the expression of unspliced viral RNA.
To investigate potential mechanisms for HIV-1 proviral latency, we generated a set of chronically HIV-1 infected and stably long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT)-transfected TE671/RD cells, and studied both their virus production and LTR-driven reporter gene expression. Established tissue culture models of retroviral latency in lymphoid and monocytoid cell lines have demonstrated that the induction of virus production is associated with a shift in HIV-1-specific mRNA from a predominance of singly and multiply spliced mRNA's to the production of full-length HIV-1 RNA. We found a similar pattern in TE671/RD cells, but in contrast to U1 and ACH2 cells, could not induce viral replication by exposure to phorbol myristate acetate (PMA) alone. We demonstrated instead that production of full-length viral RNA, viral replication, and LTR-driven CAT expression could be induced by exposure to sodium butyrate. The most proximate effect of sodium butyrate is inhibition of cellular histone deacetylase(s) which results in disruption of nucleosomes relieving one level of restriction to gene expression. Consistent with this mechanism of action, we further found that sodium butyrate's effects: (i) act synergistically with PMA and TNF-alpha; (ii) are independent of protein synthesis; (iii) do not affect the constitutively expressed creatine phosphokinase gene; (iv) do not map to a discrete sequence motif in the viral LTR; and (v) are not blocked by N-acetyl cysteine but (vi) are blocked by novobiocin, an inhibitor of cellular topoisomerase II. These data show that a similar pattern of restricted viral RNA expression exists in this nonlymphoid cellular model of HIV-1 latency. In contrast however, these results suggest that in these cells there is an additional block to viral gene expression, which is overcome with sodium butyrate. These results are discussed in the context of histone-mediated repression of HIV-1 gene expression.
Acetylcysteine↗
An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.
The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.
Amino Acid Sequence↗
Conservation of a viral RNA genome during replication and translation.
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DNA Replication↗
[Synthesis of poliomyelitis virus from viral RNA in a system of disrupted cells].
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Enterovirus C, Human↗
Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivate viral RNA synthesis.
The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.
Amino Acid Sequence↗
The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis.
Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [(3)H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication.
Acetylation↗
Specific binding of the type C viral core protein p12 with purified viral RNA.
The major viral phosphoproteins (p12) of the Rauscher murine leukemia virus (R-MuLV) and the simian sarcoma-associated virus (SSAV) bind in vitro to their homologous 70S and 35S viral RNAs. Using purified 32P-labeled RNA and 125I-labeled p12 protein, complexes that are stabilized by formaldehyde-cross-linking can be readily detected after velocity gradient centrifugation. The in vitro reconstructed ribonucleoprotein complexes are seen only with p12 proteins incubated with viral RNAs isolated from the same type C viruses; no such complexes form with heterologous protein-RNA mixtures. Homologous but not heterologous p12 molecules compete with radiolabeled p12 protein for the specific viral RNA binding sites. The competition assay permits the detection of 10 ng of viral p12 protein. The major internal protein of type C viruses (p30) does not bind to viral RNA using identical assay conditions. From the specific activities of the radiolabeled components and also by equilibrium sedimentation analysis, we estimate that fewer than 15 molecules of p12 protein bind to each molecule of viral RNA. Both the specificity and stoichiometry of the p12-RNA interactions suggest that these RNA tumor virus proteins have a regulatory role in cells.
Binding Sites↗
Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis.
Considerable controversy surrounds the impact of hepatitis C virus (HCV) protein expression on viability of host cells and regulation of the cell cycle. Both promotion of cellular proliferation and apoptosis have been observed in different experimental systems. To determine whether expression of the entire complement of HCV proteins in the context of ongoing viral RNA replication significantly alters the host cell transcriptome and cell cycle regulatory processes, we carried out high-density oligonucleotide microarray studies and analyzed cell cycle distributions and S-phase entry in Huh7 cell clones harboring selectable, full-length, replicating HCV RNAs that express the entire genotype 1b, HCV-N polyprotein, and clonally related cells in which all viral RNA was eliminated by prior treatment with alpha interferon. Oligonucleotide microarray analyses revealed only subtle, coordinated differences in the mRNA profiles of cells containing replicating viral RNA and their interferon-cured progeny, with variation between different cell clones having a greater influence on the cellular transcriptome than the presence or absence of replicating HCV RNA. Flow cytometric analysis demonstrated no significant differences in cell cycle distribution among populations of asynchronously growing cells of both types. Cell lines containing replicating viral RNA and their interferon-cured progeny were able to reenter the cell cycle similarly after transient G(1) arrest. In contrast, although viral protein expression and genome replication did not alter cell cycle control in these cells, HCV genome replication was highly dependent on cellular proliferation, with viral RNA synthesis strongly decreased in poorly proliferating, confluent, or serum-starved cells and substantially enhanced in the S phase of the cell cycle.
Cell Cycle↗