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PubMed1979

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↗
PubMed1976

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↗
PubMedDate not supplied

Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa cells.

Viral RNA was localized ultrastructurally by in situ hybridization with a biotinylated viral DNA probe and colloidal gold label in HeLa cells during infection with adenovirus type 5. Transcription was monitored by high-resolution autoradiography after short pulses with tritiated uridine. At the earliest stage of virus-induced nuclear transformation, viral RNA was restricted to the small compact fibrillar "early replicative sites" which we had previously demonstrated to be the site of viral DNA replication (Puvion-Dutilleul and Puvion, 1990b). Protease-DNase-treated sections revealed that these fibrillar masses rapidly enlarged and gave rise to a juxtaposed loose fibrillogranular structure devoid of viral RNA. Subsequently, the function of the compact fibrillar zones changed to become the sites of accumulation of single-stranded (ss) viral DNA molecules, and the contiguous new fibrillogranular zones (previously named the peripheral replicative zones, Puvion-Dutilleul and Puvion, 1990a) became not only the centers of replicating viral double-stranded DNA but also the only sites of viral RNA molecules including the elongating RNA transcripts. Pulse-chase experiments revealed an unexpected accumulation of RNA molecules in these extranucleolar regions of infected nuclei.

Adenovirus Infections, Human↗
PubMed1979

Nuclear accumulation of influenza viral RNA transcripts and the effects of cycloheximide, actinomycin D, and alpha-amanitin.

The use of virus-specific (32)P-labeled complementary DNA and (125)I-labeled virion RNA as hybridization probes has allowed us to quantitate the number of molecules of complementary RNA (cRNA) and progeny virion RNA in MDCK cells infected with influenza virus. We compared the distribution of cRNA between the nucleus and the cytoplasm in cycloheximide-treated cells to that found in untreated cells, beginning 1 h after infection. A greater percentage of the total cRNA was detected in the nucleus of the drug-treated cells at all times investigated. For the first 2 h after infection about 50% of the cRNA synthesized in the cycloheximide-treated cells was found in the nucleus. These nuclear cRNA molecules were characterized and shown to be polyadenylated transcripts of each of the genome virion RNA segments. Viral cRNA synthesis was not completely inhibited by the addition of actinomycin D at the beginning of infection, with or without the concomitant addition of cycloheximide. A large fraction (about 90%) of these cRNA sequences were detected in the nucleus. Characterization of these nuclear cRNA molecules showed that they contained polyadenylic acid and represented transcripts of both those segments coding for proteins synthesized predominantly early after infection ("early" proteins) and those virion RNA segments coding for "late" proteins. Also, in vitro translation of these cRNA molecules showed that they were functional virus mRNA's. In contrast to actinomycin D, alpha-amanitin completely inhibited cRNA synthesis when added at the beginning of infection, and addition of this drug after 1.5 h had no effect on further cRNA synthesis.

Amanitins↗
PubMed1986

Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA.

The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.

Base Sequence↗
PubMed1991

Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent.

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.

Alkylation↗
PubMed1970

DNA complementary to viral RNA in leukemic cells induced by avian myeloblastosis virus.

Nucleic acid hybridization studies were made between 71S-AMV-RNA and DNA from leukemic myeloblasts and from normal chicken cells. There was homology between the viral RNA and chicken cell DNA and to a greater extent between viral RNA and leukemic cell DNA. Leukemic cell DNA hybridized approximately twice as much viral RNA as did normal chicken DNA. Thermal melting studies showed that the viral RNA bound to normal and leukemic cell DNA consists of long polynucleotides (T(m) = 87 degrees and 92 degrees C, respectively, in 2x saline citrate). This suggests that the leukemic cells contain a DNA template of the viral RNA.

Animals↗
PubMed1974

Effect of ultraviolet light on mengovirus: formation of uracil dimers, instability and degradation of capsid, and covalent linkage of protein to viral RNA.

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.

Animals↗
PubMed1988

Isolation of poliovirus 2C mutants defective in viral RNA synthesis.

Two poliovirus mutants were isolated that contain an oligonucleotide linker insertion in the 2C-coding region of the viral genome. One, 2C-31, has a strongly temperature-sensitive phenotype and the other, 2C-32, forms small plaques on HeLa cell monolayers at all temperatures. Both mutants have a severe temperature-sensitive defect in viral RNA synthesis but little effect on the types of viral protein that are made. Temperature shift experiments showed that the 2C function is continuously required for viral RNA synthesis to proceed. The 2C mutants could be complemented in trans by mutants with mutations in other viral proteins. Protein 2C is also the locus of the guanidine resistance and dependence mutants, a drug whose action also affects viral RNA synthesis. Thus, protein 2C is one that is needed continually for viral RNA synthesis and, at least with these temperature-sensitive alleles, can be provided in trans.

Genetic Complementation Test↗
PubMed2026

PTBP1 at the host-virus interface: mechanistic roles in viral RNA translation, replication, and immune modulation.

Viruses require the involvement of host RNA binding proteins for completion of important steps of their life cycle. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein found ubiquitously which performs important regulatory functions like alternative splicing, RNA stability, RNA localization, and translation by virtue of its four RRMs and shuttling between nucleus and cytoplasm. There is increasing evidence showing that many viruses make use of such regulatory roles of PTBP1 to facilitate their gene expression and replication. This review describes the existing mechanistic knowledge about the PTBP1 functions during viral infection, paying attention to the role of PTBP1 in viral RNA translation, viral RNA genome replication, and regulation of host antiviral response. Special attention is paid to the regulation by PTBP1 of IRES-dependent translation of enteroviruses and hepatitis C virus, as well as to the PTBP1 contribution to RNA stabilization, long-distance RNA interactions, and genome cyclization of flaviviruses such as dengue virus and Japanese encephalitis virus. Recent data on the PTBP1 function in coronavirus RNA metabolism are discussed as well. Furthermore, the role of PTBP1 in being both proviral and antiviral is reviewed in terms of innate immunity signalling pathways, stress granule biology, and virus-host interaction. Finally, we will explore the possibility of PTBP1 being used as a host-directed antiviral drug target despite the hurdles in doing so considering its multifunctionality as an essential cellular RNA-binding protein.

Polypyrimidine Tract-Binding Protein↗
PubMedDate not supplied

Synthesis of long cDNA from viral RNA template.

Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

1-Propanol↗
PubMed1979

Detection of sequences in human leukemic cell DNA homologous with moloney mouse leukemia viral RNA.

DNA complementary to Moloney murine leukemia viral RNA was annealed with DNA isolated from peripheral leukocytes of twelve patients with leukemia. Six to 10% of the complementary DNA annealed to the DNA of one patient with acute myelogenous leukemia. The level of annealing of the complementary DNA to the other leukemic DNA's did not differ significantly from that to normal human spleen DNA. This result is consistent with reports of occasional positive results from other laboratories, but the significance, especially in reference to a causal role for RNA tumor viruses in human leukemia, remains unclear.

DNA↗
PubMed1992

Detection of viral RNA in experimental coxsackievirus B3 myocarditis of mice using the polymerase chain reaction.

The presence of the viral RNA in the myocardium in experimental coxsackievirus B3 myocarditis of mice was investigated using the polymerase chain reaction (PCR). Four-week-old C3H/He mice (n = 35) were inoculated with coxsackievirus B3 (Nancy strain, 10(5) plaque-forming units/mouse). We used a pair of primers, which encompass a part of the 5' end sequence of the coxsackievirus B3 genome and can also detect many enteroviral RNAs. We found that hearts were positive for the viral RNA from 2 to 21 days after virus inoculation by PCR, but negative after day 28 and in non-infected control mice (n = 5). The viral RNA were detected by PCR later than by culture. Thus, the detection of the viral RNA using enzymatic amplification is more rapid and easier and may be more useful for clinical diagnosis of viral myocarditis than conventional culture methods. However, virus persistence in the myocardium long after virus inoculation is unusual in this model.

Animals↗
PubMed1986

RNA polymerase of influenza virus. Dinucleotide-primed initiation of transcription at specific positions on viral RNA.

The priming activities of dinucleotides of all possible base sequences as to the transcription initiation by influenza virus-associated RNA polymerase were investigated. Dinucleotide ApG, complementary to positions 1-2 from the 3' termini of viral RNA segments, was the most active primer and directed the formation of ApGpC; dinucleotide GpC, complementary to positions 2-3, was also an active primer and directed the formation of either GpCpG or GpCpA; but both dinucleotides CpG and CpU, complementary to positions 3-4, were virtually inactive. These results indicate that the transcription is initiated within the first four nucleotides at the 3' termini of viral RNA. Among other dinucleotides, only those hybridizable to viral sequences at their 3'-proximal bases were partially active, implying the essential role of base pairing immediately next to the first phosphodiester bond.

Animals↗
PubMed1990

The relationship between viral RNA, myelin-specific mRNAs, and demyelination in central nervous system disease during Theiler's virus infection.

The DA strain of Theiler's murine encephalomyelitis virus (DAV) causes a chronic demyelinating disease in susceptible mouse strains. To elucidate the pathogenesis of DAV-induced demyelination, the authors investigated the spatial and chronologic relationship between virus (antigen and RNA), myelin-specific mRNAs, and demyelination in DAV-infected mice using immunohistochemistry, in situ hybridization, and slot blot hybridization analyses. In spinal cord white matter, viral RNA was detected easily in ventral root entry zones 1 to 2 weeks after infection. Viral RNA increased to maximum levels by 4 weeks after infection, which was associated with inflammation and mild demyelination. At 8 to 12 weeks after infection, when demyelination became most extensive, viral RNA was significantly decreased. Demyelination did not chronologically or spatially parallel the presence of viral RNA within the spinal cord. Decrease of myelin-specific mRNAs, including myelin-basic protein and proteolipid protein mRNAs, was observed within the demyelinating lesions with or without detectable viral RNA. These results indicate that a viral infection of white matter in the early phase of the infection initiates spinal cord disease leading to demyelination, but later an ongoing immunopathologic process contributes to the presence of extensive demyelination.

Animals↗