Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs (original) (raw)
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Characterization of Influenza Virus RNA Transcripts Synthesized in vitro
Journal of General Virology, 1979
Polyadenylated transcripts synthesized in vitro by detergent-disrupted influenza virus resemble virus mRNAs in that they possess the complement of the 3' terminus of the genome RNAs but lack sequences corresponding to the same 5' terminal region, including the homologous sequence of nucleotides I to 22. Transcription is initiated at the 3' terminus by both ApG and GpG as well as in the absence of added primer.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1986
The NS l mRNA of the influenza A virus WSN (HoN l) strain was isolated from a ceil-free transcription system, and from the cytoplasm of virus-infected HeLa cells. The 3Zp-iabeled NS t mRNA derived from the infected cell cytoplasm was characterized by the secondary enzymatic analysis of sixteen of its large or distinct RNAase Tl-resistant oligonudeotides. Several WSN strain-specific nucleotide differences from the previously-determined sequence of NS t mRNA from the PR8 (HoN t) strain of influenza A virus, were located within these sequences. The RNAase Tl-resistant oligonucleotides were placed within the primary sequence of NS t mRNA, using the PR8 strain sequence data. The resulting linear map was then used to identify NS 2 mRNA isolated from the infected cell cytoplasm, and an NS-related RNA species generated from NS l mRNA incubated in a HeLa cell-free extract.
Journal of virology, 1988
The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size t...
Pnas, 1968
There are many indications that the nucleic acid of influenza virus is not a single molecule. In 1956, Burnet' proposed that several independent genetic linkage groups exist in the virus to explain the high recombination frequency observed in genetic experiments with various strains of influenza virus. The same conclusion was reached by Hirst2 and Simpson3 who found up to 50 per cent genetic recombination in marker rescue experiments with partially inactivated influenza virus. In contrast, Newcastle disease virus (NDV), a member of the parainfluenza group of myxoviruses, failed to show genetic recombination.2 The possible existence of influenza virus RNA in several pieces is further suggested by the ready occurrence of multiplicity reactivation4 and by the preservation of some virus-specific functions such as the ability to synthesize hemagglutinin and neuraminidase after chemical inactivation of viral infectivity.5 Similarly, it was suggested2 that the von Magnus phenomenon,6 a unique characteristic of influenza virus, might indicate an uncoordinated replication of discrete segments of viral RNA. Loss of infectivity of von Magnus-type influenza virus by y-ray irradiation follows a multiple-hit curve. In contrast, inactivation of standard virus follows a single-hit curve.7 An infection caused by several "incomplete" von Magnus virus6 particles could be the reason for the observed multiple-hit inactivation curve of von Magnus virus.
Regulation of influenza virus RNA polymerase activity by cellular and viral factors
Nucleic Acids Research, 1994
An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NSI), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.
Virology, 1998
The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by ϳ74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1±90 and the other spanning residues 91±127.
Influenza A virus in vitro transcription: Roles of NS1 and NP proteins in regulating RNA synthesis
Virology, 1991
To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS,. We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication. Because NS, and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis. We showed that virion RNP complexes phosphorylated NS, but possessed no autophosphorylating activity. Soluble NP protein derived from RNP complexes did not phosphorylate NS,, but did phosphorylate casein. When NP protein was dephosphorylated, however, it no longer phosphorylated casein. We also showed that NS, was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation.
Virus Research, 1998
Mouse-adapted influenza A virus, FM-MA, interferes with the replication of wild-type strains on co-infection. The interference phenotype was previously mapped to FM-MA segment 2 encoding a mutant PB1 protein, the catalytic component of the RNA polymerase complex. To identify the point at which FM-MA interferes with wild-type A/HK/1/68 (HK), the relative levels of transcription and genome replication of the PB1, NP and M1 genes were determined for FM-MA and HK viruses in co-infected cells using RT-PCR. All stages of HK macromolecular synthesis (primary and secondary transcription, genomic RNA, complementary RNA and protein synthesis) were suppressed relative to FM-MA. Infection with HK virus alone resulted in the accumulation of similar or greater amounts of RNA at late times post-infection relative to FM-MA thus indicating that the presence of FM-MA specifically compromised HK transcription and replication in co-infected cells. However early in infection FM-MA was ten times more active in mRNA transcription than HK or its parental strain FM. FM-MA's ability to interfere was primarily due to an increased capacity for primary transcription. FM-MA genomes were also selectively assembled into progeny virus from cells co-infected with HK and FM-MA, a step which was distinct from the capacity for enhanced RNA synthesis. This suggests that interference of HK growth by FM-MA in mixed infections results from two distinct events: a preferential synthesis of FM-MA-specific macromolecules which is then augmented by a preferential assembly of FM-MA genomes.