No abstract

Influenza is a contagious respiratory illness in humans caused by influenza A and B viruses that results in an annual average of 36,000 deaths in the United States alone (www.cdc. gov/Flu). Influenza A viruses, infecting birds and mammals, periodically cause widespread pandemics, the most deadly of which was the 1918 "Spanish Flu" pandemic that claimed an estimated 50 million lives (1). In recent years, the appearance of new H5N1 and H1N1 influenza A viruses, known as "avian flu" and "swine flu," respectively, has fueled fears of an impending deadly influenza pandemic in the 21st century. Moreover, the rapid emergence of influenza virus strains resistant to current antiviral drugs directed against influenza A neuraminidase and M2 ion channel accentuate the need for the development of new classes of influenza antivirals. Toward this end, several recent structural and functional studies of influenza proteins have illuminated new potential drug targets in influenza (2, 3).One such target is the non-structural protein 1 of influenza A, NS1A, a key multifunctional virulence factor produced in the host infected cell that plays a critical role in evading the host antiviral response (4). This highly conserved protein is composed of two domains, the 73-residue N-terminal doublestranded RNA-binding domain (RBD) 4 and the C-terminal (residues 86-end) effector domain (ED), joined by a flexible linker (Fig. 1). By binding non-specifically to dsRNA, the N-terminal RBD functions primarily to inhibit the interferon-induced 2Ј-5Ј oligonucleotide A synthetase/RNase L pathway (5). The C-terminal ED binds a plethora of host cellular proteins, including the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) (6, 7), the p85␤ subunit of phosphatidylinositol 3-kinase (PI3K) (8, 9), protein kinase R (PKR) (10, 11), and human tripartite motif 25, TRIM25, the E3 ubiquitin ligase of the retinoic acid-inducible gene I (RIG-I) (12, 13). All of these activities of NS1A ultimately contribute to the ability of influenza virus to suppress host responses to infection, including interferon production and apoptosis, critical to the life cycle of the virus. The recent developments of novel NS1 antagonists (14) and attenuated influenza viruses containing * This work was supported, in whole or in part, by National Institutes of Health Grant U54-GM094597 through the NIGMS Protein Structure Initiative (to G. T. M.) and National Institutes of Health Grants U01 AI074497 (to G. T. M. and R. M. K.) and R01 AI11772 (to R. M. K. 3 To whom correspondence may be addressed. E-mail: guy@cabm.rutgers.edu. 4 The abbreviations used are: RBD, RNA-binding domain; ED, effector domain; HSQC, heteronuclear single quantum coherence; TROSY, transverse relaxation optimized spectroscopy; SUMO, small ubiquitin-like modifier.

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex.

Summary Influenza A virus RNA synthesis is catalyzed by the viral polymerase comprised of the PA, PB1 and PB2 proteins. We show that the host DDX21 RNA helicase restricts influenza A virus by binding PB1 and inhibiting polymerase assembly, resulting in reduced viral RNA and protein synthesis. Later during infection, the viral NS1 protein overcomes this restriction by binding to DDX21 and displacing PB1. DDX21 binds to a region of the NS1 N-terminal domain that also participates in other critical functions. A virus mutant whose NS1 protein is unable to bind DDX21 exhibits reduced viral protein synthesis at both late and early times of infection, a phenotype converted to wild-type upon DDX21 knockdown. As sequential interaction of PB1 and NS1 with DDX21 leads to temporal regulation of viral gene expression, influenza A virus likely uses the DDX21-NS1 interaction not only to overcome restriction but also to regulate the viral life cycle.