Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs.

Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.

The influenza virus NS1 protein forms multimers in vitro and in vivo.

The NS1 protein of the influenza A virus inhibits both the nuclear export of mRNA and pre-mRNA splicing. Two functional domains, an RNA-binding domain and an effector domain, have been identified in this protein. Here we demonstrate that the NS1 protein exists as a dimer in vitro both in the absence of its RNA target and when it is bound to a specific RNA target, U6 snRNA. This indicates that it is most likely the dimer that binds to the RNA target. Mutational analysis indicated that the RNA-binding and dimerization domains are coincident. Multimerization also occurs in vivo, as assayed using the yeast two-hybrid system. In contrast to the situation in vitro, multimerization in vivo was mediated by not only the RNA-binding domain but also the effector domain. This suggests that multimerization in vivo involves a cellular protein cofactor that bridges more than one NS1 protein molecule together via their effector domains.

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA.

Influenza virus unspliced NS1 mRNA, like retroviral pre-mRNAs, is efficiently exported from the nucleus and translated in the cytoplasm of infected cells. With human immunodeficiency virus (HIV), the transport of viral pre-mRNAs is facilitated by the viral Rev protein. We tested the possibility that the influenza virus NS1 protein, a nuclear protein that is encoded by unspliced NS1 mRNA, has the same function as the HIV Rev protein. Surprisingly, using transient transfection assays, we found that rather than facilitating the nucleocytoplasmic transport of unspliced NS1 mRNA, the NS1 protein inhibited the transport of NS2 mRNA, the spliced mRNA generated from NS1 mRNA. The efficient transport of NS2 mRNA from the nucleus to the cytoplasm occurred only when the synthesis of the NS1 protein was abrogated by amber mutations. The NS1 protein down-regulated the export of NS2 mRNA whether or not it was generated by splicing, indicating that the NS1 protein acted directly on transport. Actinomycin D chase experiments verified that the NS1 protein acted on the transport and not on the differential stability of NS2 mRNA in the nucleus as compared to the cytoplasm. In addition, the NS1 protein inhibited the transport of NS1 mRNA itself, which contains all of the sequences in NS2 mRNA, particularly when NS1 mRNA was released from the splicing machinery by mutating its 3'-splice site. Our results indicate that the NS1 protein-mediated inhibition of transport requires sequences in NS2 mRNA. The transport of the viral PB1 protein, nucleocapsid protein, hemagglutinin, membrane protein, and M2 mRNAs was not affected by the NS1 protein. When the NS2 mRNA sequence was covalently attached to the PB1 mRNA, the transport of the chimeric mRNA was inhibited by the NS1 protein. Our results identify a novel function of the influenza virus NS1 protein and demonstrate that post-transcriptional control of gene expression can also occur at the level of the nucleocytoplasmic transport of a mature, spliced mRNA.

Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.

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.

Construction of full-length cDNA copies of viral double-stranded RNA.

A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end. The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14. Two of these were complete at the 3' end, and one lacked a single base here. Of these four clones, one also retained the XbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.

Initiation by the yeast viral transcriptase in vitro.

All double-stranded RNA viruses have capsid-associated transcriptase activities. In the yeast viruses, as in reovirus, transcription appears to be the first stage of replication. We have found that the yeast viral transcriptase initiates RNA transcription in vitro and that the resultant plus strand RNA has the 5' terminus ppGp. No pre-existing primers are normally utilized in vitro. Like other double-stranded RNA viruses of eucaryotes, the yeast viruses have a primer-independent capsid-associated transcriptase. Unlike these viruses of higher eucaryotes, the yeast viruses synthesize uncapped mRNAs. Viral particles with only a single major capsid polypeptide are active in transcription and replication, while reovirus particles active in transcription have 5 or 6 polypeptides.

Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus.

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.

Conservative replication and transcription of Saccharomyces cerevisiae viral double-stranded RNA in vitro.

All double-stranded RNA viruses have capsid-associated RNA polymerase activities. In the reoviruses, the transcriptase synthesizes the viral plus strand in a conservative mode and the replicase synthesizes the viral minus strand, again conservatively. In bacteriophage phi 6 and in some fungal viruses, the transcriptase activity is semiconservative, acting by displacement synthesis. In this work we demonstrate Saccharomyces cerevisiae viral RNA replication in vitro for the first time and, using more sensitive techniques than those previously used, show that both the transcriptase and the replicase appear to act conservatively, like those of reovirus. There is therefore clearly no universal life cycle for the double-stranded RNA viruses.