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...