Robert M. Krug

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.

Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery.

Influenza A virus NS1 protein (NS1A protein) via its effector domain targets the poly(A)‐binding protein II (PABII) of the cellular 3′‐end processing machinery. In vitro the NS1A protein binds the PABII protein, and in vivo causes PABII protein molecules to relocalize from nuclear speckles to a uniform distribution throughout the nucleoplasm. In vitro the NS1A protein inhibits the ability of PABII to stimulate the processive synthesis of long poly(A) tails catalyzed by poly(A) polymerase (PAP). Such inhibition also occurs in vivo in influenza virus‐infected cells, where the NS1A protein via its effector domain causes the nuclear accumulation of cellular pre‐mRNAs which contain short (∼12 nucleotide) poly(A) tails. Consequently, although the NS1A protein also binds the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF), 3′ cleavage of some cellular pre‐mRNAs still occurs in virus‐infected cells, followed by the PAP‐catalyzed addition of short poly(A) tails. Subsequent elongation of these short poly(A) tails is blocked because the NS1A protein inhibits PABII function. Nuclear–cytoplasmic shuttling of PABII, an activity implicating this protein in the nuclear export of cellular mRNAs, is also inhibited by the NS1A protein. In vitro assays suggest that the 30 kDa CPSF and PABII proteins bind to non‐overlapping regions of the NS1A protein effector domain and indicate that these two 3′ processing proteins also directly bind to each other.

RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids

The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2, which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5′‐ and 3′‐terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U‐substituted RNA and UV crosslinking, we demonstrate that the 5′ and 3′ sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA‐binding sites. The 5′ sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5′‐capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3′ sequence of vRNA, via a ribonucleoprotein 1 (RNP1)‐like motif, amino acids 249–256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA‐chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.

Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5′ splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5′ splice site. Second, utilization of the weak M2 5′ splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine‐rich splicing enhancer sequence that is located in the 3′ exon of M1 mRNA. We demonstrate that activation of the M2 5′ splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.

The regulation of export of mRNA from nucleus to cytoplasm.

The past year has been marked by the discovery that the influenza virus NS1 protein belongs to the group of viral proteins that regulate the nuclear export of mRNA. This protein, like other viral proteins in this group, such as the Rev protein of human immunodeficiency virus 1 (HIV-1) and the complex of two adenovirus early proteins, has the potential to provide insights into the poorly understood process of the nuclear export of mRNA.

Translational control in influenza virus-infected cells

Influenza virus type A has been shown to establish a translational control system such that during infection there is a dramatic inhibition of host cell protein synthesis and viral mRNAs are selectively and efficiently translated. The following review summarizes the complex strategies employed by influenza to accomplish these goals. These include: (i) preventing newly made cellular mRNAs from entering the cytoplasm of infected cells; (ii) inhibiting the initiation and elongation steps of translation of preexisting cellular mRNAs; (iii) possessing RNAs with structural features which enhance translation; (iv) encoding mechanisms to downregulate the interferon induced protein kinase thus allowing overall protein synthesis levels to remain high.

Influenza Viral RNA Transcription

Influenza viral messenger RNA (mRNA) synthesis is initiated by cap 1 (m7GpppNm)-containing primers generated from heterologous RNAs. We have elucidated the functions and movements of the three viral P (PB1, PB2 and PA) proteins during viral mRNA synthesis catalyzed by purified virion nucleocapsids in vitro. Viral mRNA synthesis occurs in the nucleus of infected cells, leading to: (i) disruption of the metabolism of host cell RNA polymerase II transcripts; and (ii) the modification (methylating of internal A residues and splicing) of viral mRNAs by host nuclear enzymes. In the infected cell, another type of viral transcript, full-length transcripts, are synthesized, which, unlike the viral mRNAs, are initiated without a primer and contain a copy of the last 17–22 nucleotides at the 5′ ends of the virion RNAs. We describe the development of an in vitro system that catalyzes the synthesis of these transcripts, the presumed templates for virion RNA synthesis. Finally, we will show that influenza virus establishes a translational system that selectively translates viral and not host mRNAs.