The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.

Abstract

We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NSI protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral nsl mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in nsl mRNA.[Key Words: Influenza virus NS1 protein; pre-mRNA splicing; spliceosome; U6 snRNA] Received May 12, 1994; revised version accepted June 16, 1994.Eukaryotic pre-mRNAs synthesized by nuclear RNA polymerase II undergo a series of post-transcriptional processes. Two of these processes are pre-mRNA splicing and mRNA export from the nucleus, both of which can be regulated (Green 1989;Krug 1993). Alternative splicing, whereby different mRNAs are generated from a single pre-mRNA, has been shown to be regulated by proteins that bind to specific regions of the pre-mRNA precursor. In many instances it has been shown that specific proteins act on specific pre-mRNA targets to regulate their alternative splicing. For example, in the Drosophila sex determination pathway, a specific protein(s)--sex lethal protein or the complex of the Tra and Tra2 proteins--binds to a specific site on a particular premRNA to either suppress or activate, respectively, the usage of a 3' splice site (Baker 1989;Inoue et al. 1990;Bell et al. 1991;Tian and Maniatis 1992).The nuclear export of unspliced pre-mRNAs and spliced mRNAs can also be regulated by specific proteins. Usually unspliced pre-mRNAs are not exported from the nucleus, apparently at least in part because the pre-mRNAs are efficiently committed to spliceosome formation in the nucleus (Legrain and Rosbash 1989). However, this process is overcome in the case of the pre-mRNAs of human immunodeficiency virus-1 (HIV-1) and other lentiviruses by the action of the virus-encoded Rev (or Rev-like) protein, which binds to a specific RNA sequence (RRE, or Rev responsive element) in the viral pre-mRNAs and facilitates their nuclear export (Hadzopoulou-Cladaras et al. 1989; Hanly et al. 1989;Malim et al. 1989). The influenza virus NS1 protein has the opposite effect on the nuclear export of mRNA: It inhibits the transport of mRNAs (Alonso-Caplen et al. 1992;Fortes et al. 1994). The NS1 protein binds to the poly(A) sequence at t...

The 3′-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo

“…The effector domain of the NS1A protein is sufficient for the inhibition of 39-end processing of cellular pre-mRNAs in vivo Previous studies have established that the effector domain of the NS1A protein is required for the inhibition of 39-end processing of cellular pre-mRNAs (Nemeroff et al+, 1998;Chen et al+, 1999)+ Both the 30-kDa CPSF and PABII proteins bind to the effector domain, and deletion of the effector domain eliminates the ability of the NS1A protein to inhibit 39-end processing+ A requirement for the RNA-binding domain, as well as the effector domain, in the inhibition of 39-end processing was suggested by early mutagenesis experiments )+ Mutations in the RNA-binding domain as well as in the effector domain of the NS1A protein resulted in the loss of the ability of the NS1A protein to block the nuclear export of cellular mRNAs+ This export block has been shown to result from the inhibition of 39-end processing of cellular pre-mRNAs (Nemeroff et al+, 1998;Chen et al+, 1999)+ However, the mutant NS1A proteins used in these studies are inactive in both RNA binding and dimerization (Lu et al+, 1994;Qiu & Krug, 1994;Qian et al+, 1994;Nemeroff et al+, 1995;Wang et al+, 1999)+ Because the loss of dimerization is a profound change in the structure of the NS1A protein, the effector domain as well as the RNA-binding domain may be inactivated in such mutant proteins+ Consequently, to determine whether the RNA-binding domain is required for the inhibition of 39-end processing, we used a different mutant NS1A protein that was designed based on the results obtained with the RNAbinding fragment of the protein (amino acids 1-73) (Wang et al+, 1999)+ Only two amino acids, the arginine at position 38 and the lysine at position 41, in the RNAbinding fragment are required for efficient RNA binding, and these two amino acids are not required for dimerization+ Accordingly, we replaced these two basic amino acids in the full-length NS1A protein with alanines, and determined whether the resulting mutant NS1A protein (38/41 mutant) dimerizes and possesses RNA-binding activity+ As a control, we replaced the two arginines at positions 35 and 46 with alanines (35/46 mutant), because these replacements in the RNA-binding fragment result in a loss of dimerization as well as of RNAbinding activity (Wang et al+, 1999)…”

“…In the present study, we use the NS1 protein of influenza A virus (NS1A protein) to elucidate the role of 39-end processing in the splicing of the 39 terminal intron of mammalian pre-mRNAs in vivo+ This viral protein contains two functional domains: an RNA-binding/ dimerization domain at the amino terminus and an effector domain in the carboxy half Wang & Krug, 1996)+ The effector domain interacts with two essential components of the machinery for the 39-end processing of cellular pre-mRNAs: the 30-kDa subunit of CPSF and poly(A)-binding protein II (PABII) (Nemeroff et al+, 1998;Chen et al+, 1999)+ The consequent inhibition of CPSF and PABII function efficiently blocks the 39-end processing of cellular pre-mRNAs in both transfected cells and influenza virus-infected cells, and, as a result, cellular mRNAs are not exported from the nucleus (Nemeroff et al+, 1998;Chen et al+, 1999)+ In addition, as established by transfection experiments, the NS1A protein inhibits the in vivo splicing of premRNAs containing a single intron (Fortes et al+, 1994;Lu et al+, 1994)+ We demonstrate that inhibition of the splicing of singleintron pre-mRNAs results from inhibition of 39-end processing, thereby establishing that 39-end processing is required for the splicing of 39 terminal introns in vivo+ By employing the NS1A protein that causes a global suppression of 39-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA+ In addition, this strategy enabled us to establish that the function of a particular 39-end processing factor, namely CPSF, is required for the splicing of single intron pre-mRNAs in vivo+…”

Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs

“…Cis-acting elements that enhance the cytoplasmic accumulation of unspliced or incompletely spliced RNAs are also found in hepatitis B virus (Huang & Liang, 1993 ;Huang & Yen, 1994) and herpes simplex virus type 1 (Greenspan & Weissman, 1985 ;Liu & Mertz, 1995). In influenza virus-infected cells nucleocytoplasmic transport of viral and cellular RNAs and pre-mRNA splicing are regulated by the viral NS1 protein (Alonso-Caplen et al, 1992 ;Chen et al, 1999 ;Fortes et al, 1994 ;Lu et al, 1994).…”

Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K

“…Two functional domains have been identified in the NS1 protein: an RNA-binding domain near the N terminus and an effector domain in the C terminus . The RNA-binding activity of the NS1 protein correlates to its ability to inhibit cellular pre-mRNA splicing (Lu et al, 1994;Qiu et al, 1995). It is also linked to the ability to counteract cellular alpha/beta interferon functions efficiently (Garcia-Sastre et al, 1998) by inhibiting the activation of the protein kinase PKR (Lu et al, 1995;Hatada et al, 1999) and transcription factors NF-kB, IRF-3 and IRF-7 (Talon et al, 2000;Wang et al, 2000).…”