Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.The influenza A virus contains eight segmented and negative-stranded RNAs as its genome. The viral RNAs (vRNA) are associated with the viral RNA-dependent RNA polymerase subunits (PB1, PB2, and PA) and nucleoprotein (NP), forming structurally distinct viral ribonucleoprotein (vRNP) complexes (36). The vRNP complex is a basic unit for active transcription and replication. Transcription and replication of vRNA occur in the nuclei of infected cells. The PB1 subunit plays a central role in the catalysis of the polymerization of the RNA chain. It contains amino acid motifs that are common to RNA-dependent RNA polymerases and RNA-dependent DNA polymerases (2). The PB2 subunit is required for the transcription of vRNA. It binds to the methylated cap-1 structure of host RNAs, and the capped oligonucleotide RNA is endonucleolytically cleaved by the PB1 subunits (8, 15). The resultant 10-to 13-nucleotide-long capped RNA fragment serves as a primer for viral mRNA synthesis. Genetic analyses suggest that the PA subunit is required for vRNA replication (14). The PA subunit induces a generalized proteolytic process (23, 34), and it is involved in the assembly of the polymerase subunits (13).In negative-strand RNA viruses, RNA-dependent RNA polymerases are present in the virion. Purified vRNP complexes or RNA polymerases catalyze transcription from vRNA in vitro; however, the vRNP complexes alone are not sufficient for genome replication or for the efficient transcription of viral RNAs. Some of the paramyxoviruses and rhabdoviruses have been shown to require host factors for efficient RNA synthesis in vitro. Tubulin is involved in the transcription of vesicular stom...

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Efficient transcription and replication of the influenza virus genome are dependent upon host-derived factors. Using an in vitro RNA synthesis system, we have purified and identified Hsp90 as one of the host factors that stimulate viral RNA polymerase activity. Hsp90 interacted with the PB2 subunit of the viral RNA polymerase through the amino-terminal chaperone domain and the middle region containing a highly acidic domain. The acidic middle region was also responsible for its stimulatory activity. We found that a portion of Hsp90 is re-localized to the cell nucleus after viral infection. A PB2 fragment containing a Hsp90 binding domain inhibited viral gene expression in a dominant-negative manner. These results suggest that Hsp90 is a host factor for the influenza virus RNA polymerase.Influenza A virus belongs to the Orthomyxoviridae family, and its genome consists of eight segmented, single-stranded RNA of negative polarity (1). The transcription promoter and the replication signal of the viral genome exist at the 3Ј and 5Ј termini of each of the eight segments. Components associated with ribonucleoprotein complexes (vRNP) 1 purified from virions are the minimum factors required for primary transcription. The genome RNA forms vRNP with the viral RNA polymerases consisting of three subunits, PB2, PB1, and PA (2), and nucleocapsid protein (NP). Transcription of the influenza virus genome is initiated with host-derived oligo RNA containing a cap structure. PB2 contains cap recognition domains at its carboxyl-terminal region. The capped RNA bound to PB2 is cleaved by the PB1 subunit 10 -15 bases downstream from the 5Ј end (2-4), and the capped RNA fragment serves as a primer for viral mRNA synthesis catalyzed by PB1 (5). Elongation of the RNA chain proceeds until the polymerase reaches a polyadenylation signal consisting of 5-7 uracil (U) residues located near the 5Ј terminal region of the vRNA (6). The viral RNA polymerase polyadenylates the nascent RNA chain possibly by a slippage mechanism at the U-stretch (7). Replication of the vRNA is thought to take place by a primer-independent, twostep reaction, namely the complementary RNAs (cRNA) are first synthesized from vRNA templates, and then the progeny vRNAs are amplified from cRNA templates. Genetic analyses suggest that PA participates in the replication process (8). However, vRNP complexes isolated from virions are incapable of catalyzing replication reactions.A variety of host proteins have been identified as factors involved in the regulation of the RNA synthesis of viral genomes of Paramyxoviridae, the genome of which contains nonsegmented and single-stranded RNA of negative polarity. Tubulin, an acidic cytoplasmic structural protein, is one of the host factors for RNA synthesis of the measles virus, VSV, and Sendai virus genomes (9, 10). RNA synthesis of these viral genomes is catalyzed by viral RNA polymerases consisting of L and P subunits. Tubulin interacts with L protein, a catalytic subunit of the viral RNA polymerase, and is present in isolated tr...

Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.The genome of influenza A virus consists of eight singlestranded RNA segments of negative polarity. These viral RNA (vRNA) segments exist as ribonucleoprotein (vRNP) complexes with nucleocapsid proteins (NP) and viral RNA polymerases as components. Each RNA segment contains highly conserved 3Ј-and 5Ј-terminal untranslated regions which function as regulatory signals for transcription and replication of the genome. The partially hybridized terminal regions have been referred to as panhandle (9), fork, hook, and corkscrew (14,15,25,44) forms. In vRNP complexes prepared from purified virions, viral RNA polymerase is found bound to the panhandle region (33), and NP is bound to vRNA such that each NP monomer occupies approximately 20 nucleotides (6, 56).Studies using the vRNP isolated from virions have revealed that viral RNA polymerase and NP are essential for transcription (3,21,24). The viral RNA polymerase consists of PB2, PB1, and PA subunits and is capable of initiating primerdependent RNA synthesis (20, 27). However, for synthesis of full-length RNA, NP is required (21,22). Transcription is initiated by recognition by PB2 of the cap structure of nuclear pre-mRNA. PB2 truncates the capped RNA at 10 to 13 bases downstream from the 5Ј end (3, 42). After the capped oligonucleotide is cleaved, it serves as a primer for viral mRNA synthesis catalyzed by PB1 (17). Elongation of the RNA chain proceeds until the polymerase reaches a polyadenylation signal, consisting of five to seven U residues located near the 5Ј-terminal region of the vRNA (29). The viral RNA polymerase polyadenylates the nascent RNA chain, possibly by a slippage mechanism at the U stretch (43). Replication of vRNA is a primer-independent two-step reaction: first, cRNAs are synthesized from vRNA templates; and second, the progeny vRNAs are amplified from cRNA templates. Genetic analyses suggest that PA participates in the replication process. However, vRNP co...

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The influenza virus RNA-dependent RNA polymerase is capable of initiating replication but mainly catalyzes abortive RNA synthesis in the absence of viral and host regulatory factors. Previously, we reported that IREF-1/minichromosome maintenance (MCM) complex stimulates a de novo initiated replication reaction by stabilizing an initiated replication complex through scaffolding between the viral polymerase and nascent cRNA to which MCM binds. In addition, several lines of genetic and biochemical evidence suggest that viral nucleoprotein (NP) is involved in successful replication. Here, using cell-free systems, we have shown the precise stimulatory mechanism of virus genome replication by NP. Stepwise cell-free replication reactions revealed that exogenously added NP free of RNA activates the viral polymerase during promoter escape while it is incapable of encapsidating the nascent cRNA. However, we found that a previously identified cellular protein, RAF-2p48/NPI-5/UAP56, facilitates replication reaction-coupled encapsidation as an NP molecular chaperone. These findings demonstrate that replication of the virus genome is followed by its encapsidation by NP in collaboration with its chaperone.The genome of influenza type A viruses consists of eightsegmented and single-stranded RNAs of negative polarity. Transcription from the viral RNA (vRNA) genome is initiated using the oligonucleotide containing the cap-1 structure from cellular pre-mRNAs as a primer, whereas genome replication is primer independent and generates full-length vRNA through cRNA (full-sized complementary copy of vRNA) (reviewed in reference 17). Generally, each viral DNA or RNA genome is not present as a naked form but as a complex with viral basic proteins. The influenza virus genome exists as a ribonucleoprotein (termed vRNP) complex with nucleoprotein (NP), one of the basic viral proteins, and viral RNA-dependent RNA polymerases consisting of three subunits (PB1, PB2, and PA). NP binds single-stranded RNA without sequence specificity and is required for maintaining the RNA template in an ordered conformation suitable for viral RNA synthesis and packaging into virions (6,23,34). In the case of Mononegavirales, nonsegmented and negative-stranded RNA viruses, it is proposed that the nucleocapsid (N) protein forms a trimeric complex with the viral RNA polymerase large (L) protein and phosphoprotein (P) to form a replicase complex to produce the progeny viral genome with concomitant encapsidation of nascent RNA by N protein and that encapsidation is mediated by the chaperone activity of P protein (2,7,14,24). In the case of influenza virus, it is also postulated that NP might regulate the viral polymerase function and encapsidate the virus genome through its interaction with PB1 and/or PB2 (1, 23). Genetic analyses suggest that NP participates in the replication process (15). Recently, it was also shown that NP that is saturated with single-stranded DNA (ssDNA), resulting in the lack of RNA binding activity, stimulates virus genome replication from ...

In polarized epithelial cells, influenza A virus hemagglutinin (HA) and neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical plasma membrane for viral assembly and budding. Previous studies have indicated that the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA are required for association with lipid rafts, but the raft dependencies of their apical targeting are controversial. Here, we show that coexpression of HA with NA accelerated their apical targeting through accumulation in lipid rafts. HA was targeted to the apical plasma membrane even when expressed alone, but the kinetics was much slower than that of HA in infected cells. Coexpression experiments revealed that apical targeting of HA and NA was accelerated by their coexpression. The apical targeting of HA was also accelerated by coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and the deletion of the CT in HA and NA that reduced their association with lipid rafts abolished the acceleration of their apical transport, indicating that the lipid raft association is essential for efficient apical trafficking of HA and NA. An in situ proximity ligation assay (PLA) revealed that HA and NA were accumulated and clustered in the cytoplasmic compartments only when both were associated with lipid rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings. We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism of the accelerated apical transport of HA and NA via clustering of lipid rafts. IMPORTANCELipid rafts serve as sites for viral entry, particle assembly, and budding, leading to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase (NA) of influenza virus, key players for particle assembly, contain determinants for apical sorting and lipid raft association. However, it remains to be elucidated how lipid rafts contribute to the apical trafficking and budding. We investigated the relation of lipid raft association of HA and NA to the efficiency of apical trafficking. We show that coexpression of HA and NA induces their accumulation in lipid rafts and accelerates their apical targeting, and we suggest that the accelerated apical transport likely occurs by clustering of lipid rafts at the TGN. This finding provides the first evidence that two different raft-associated viral proteins induce lipid raft clustering, thereby accelerating apical trafficking of the viral proteins.

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