Influenza Virus NS1 Research Articles

The expression of canine cardiac phospholamban in heterologous systems

A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein. Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli. Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart. Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments.

Inhibitory effects of vesicular stomatitis virus on cellular and influenza viral RNA metabolism and protein synthesis

Infection with vesicular stomatitis virus (VSV) results in the rapid inhibition of cellular macromolecular synthesis, including transcription, translation, and maturation of the U1 and U2 snRNPs. Unlike infection with VSV, influenza virus infection did not result in the inhibition of either the processing of Ul and U2 snRNAs or the assembly of the RNPs. Although influenza virus relies on the cellular splicing apparatus to generate viral mRNAs, the maturation of snRNPs was inhibited during double infections with VSV. However, this inhibition of snRNP maturation had no effect on the splicing of a cellular pre mRNA in extracts prepared from VSV-infected HeLa cells. Thus, the effects of VSV on the processing and assembly of snRNPs appear to involve virus-specific functions and unique cellular targets. Coinfection with VSV and influenza virus resulted in the dramatic inhibition of influenza virus transcription; polyadenylated mRNAs corresponding to the influenza virus NP and NS1 proteins could not be detected by Northern blot analysis. However, reduced levels of the influenza virus replicative templates were still synthesized during double infection. Coinfection with VSV also resulted in the inhibition of influenza viral mRNA translation, even when superinfection with VSV was delayed until 3 or 6 hr after influenza virus infection. VSV required at least 2 hr to affect the inhibition of translation; this corresponded to the time after VSV infection when inhibition of cellular protein synthesis was evident. These results demonstrate that, in contrast to adenovirus, the VSV-mediated inhibition of cellular macromolecular synthesis maybe effective against influenza virus.

A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the U1, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' and branch point of NS1 and mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 is controlled in infected cells.

A Block in Mammalian Splicing Occurring after Formation of Large Complexes Containing U1, U2, U4, U5, and U6 Small Nuclear Ribonucleoproteins

The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the U1, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' and branch point of NS1 and mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 is controlled in infected cells.

Two nuclear location signals in the influenza virus NS1 nonstructural protein.

The NS1 protein of influenza A virus has been shown to enter and accumulate in the nuclei of virus-infected cells independently of any other influenza viral protein. Therefore, the NS1 protein contains within its polypeptide sequence the information that codes for its nuclear localization. To define the nuclear signal of the NS1 protein, a series of recombinant simian virus 40 vectors that express deletion mutants or fusion proteins was constructed. Analysis of the proteins expressed resulted in identification of two regions of the NS1 protein which affect its cellular location. Nuclear localization signal 1 (NLS1) contains the stretch of basic amino acids Asp-Arg-Leu-Arg-Arg (codons 34 to 38). This sequence is conserved in all NS1 proteins of influenza A viruses, as well as in that of influenza B viruses. NLS2 is defined within the region between amino acids 203 and 237. This domain is present in the NS1 proteins of most influenza A virus strains. NLS1 and NLS2 contain basic amino acids and are similar to previously defined nuclear signal sequences of other proteins.

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli

The complete sequences of the SV40 agnogene ( LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 μg/ml in the case of NS1A-LP1 to approx. 5 μg/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with M rs expected for isolated NS1A and LP 1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.