Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 A crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.

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.

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Of the several hundred proteins induced by interferon (IFN) a/b, the ubiquitin-like ISG15 protein is one of the most predominant. We demonstrate the novel way in which the function of the ISG15 protein is inhibited by in¯uenza B virus, which strongly induces the ISG15 protein: a speci®c region of the in¯uenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of ISG15 to its target proteins both in vitro and in infected cells. We identify UBE1L as the E1 enzyme that catalyzes the ®rst activation step in the conjugation of ISG15, and show that the NS1B protein inhibits this activation step in vitro. In¯uenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection. We discuss the likely basis for these different strategies.

Our interest in ISG15 originated in the course of experiments to elucidate the function of the NS1B protein of influenza B virus. We found that the NS1B protein binds ISG15 and inhibits its conjugation (6), indicating that ISG15 conjugation is likely to be an important part of the IFN-␣͞␤-induced antiviral response. However, it was not evident how ISG15 conjugation might serve such a role. To address this issue and to elucidate the function of ISG15 conjugation, we first identified the E1 and E2 enzymes in the ISG15 conjugation pathway as Ube1L and UbcH8, respectively, both of which are induced by IFN-␣͞␤ (6, 7). These findings enabled us to develop a system for a proteomics-based identification of ISG15 target proteins, which is described in the present study.We used this system to identify a large number (158) of ISG15 modified proteins in IFN-␤-treated human (HeLa) cells. The identity of these ISG15 target proteins provides insights into the function of ISG15 modification. Several of the targets are IFN-␣͞ ␤-induced antiviral proteins, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed human proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling͞ polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions and may have a role in regulating transcription and pre-mRNA splicing during the IFN-␣͞␤ response. Thus, by targeting this wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-␣͞␤. Materials and MethodsPlasmids. Plasmids containing the following PCR-generated reading frames were inserted into pcDNA3 vectors: Ube1L, UbcH8, His 6 -HA-ISG15, and His 6 -3xFLAG-ISG15. All of the cDNAs used for verifying ISG15 target proteins, except maspin, were generated by PCR by using a Human Leukocyte Matchmaker cDNA library (Clontech). The template for amplifying maspin was pEF-Maspin, provided by Zhang Min (Baylor School of Medicine, Houston). For the expression of V5-tagged target proteins, two modified pcDNA3 vectors containing the V5 epitope were constructed. The original BamHI site of pcDNA3 was eliminated and replaced by the V5 sequence followed by either a BamHI site (pcDNA3-V5-Bam) or a NotI site (pcDNA3-V5-Not). The PCR-generated reading frames for maspin, PTB-1, and thioredoxin reductase-1 (TrxR1) were cloned into pcDNA3-V5-Bam as BglII-BglII, BglII-R1, and BamH-R1 fragments, respectively. The PCR-generated reading frames for Hsp60 and moesin were cloned into pcDNA3-V5-Not as Not-XbaI and Not-EcoRI fragments, respectively. For the expression of 3xFLAG-RIG-I, its PCR-generated reading frame was inserted into the pCMV10 vector (Sigma).Purification of ISG15 Conjugates. HeLa cells in each of five 150-mm culture dishes (total of 10 8 cells) were transfected by using Fugene 6 ...