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...

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...

Influenza A virus (IAV) membrane proteins hemagglutinin (HA) and neuraminidase (NA) are determinants of virus infectivity, transmissibility, pathogenicity, host specificity, and major antigenicity. HA binds to a virus receptor, a sialoglycoprotein or sialoglycolipid, on the host cell and mediates virus attachment to the cell surface. The hydrolytic enzyme NA cleaves sialic acid from viral receptors and accelerates the release of progeny virus from host cells. In this study, we identified a novel function of HA and NA as machinery for viral motility. HAs exchanged binding partner receptors iteratively, generating virus movement on a receptor-coated glass surface instead of a cell surface. The virus movement was also dependent on NA. Virus movement mediated by HA and NA resulted in a three to four-fold increase in virus internalisation by cultured cells. We concluded that cooperation of HA and NA moves IAV particles on a cell surface and enhances virus infection of host cells.

Influenza viruses infect vertebrates, including mammals and birds.Influenza virus reverse-genetics systems facilitate the study of the structure and function of viral factors. In contrast, less is known about host factors involved in the replication process. Here, we developed a replication and transcription system of the negativestrand RNA genome of the influenza virus in Saccharomyces cerevisiae, which depends on viral RNAs, viral RNA polymerases, and nucleoprotein (NP). Disruption of SUB2 encoding an orthologue of human RAF-2p48/UAP56, a previously identified viral RNA synthesis stimulatory host factor, resulted in reduction of the viral RNA synthesis rate. Using a genome-wide set of yeast single-gene deletion strains, we found several host factor candidates affecting viral RNA synthesis. We found that among them, Tat-SF1, a mammalian homologue of yeast CUS2, was a stimulatory host factor in influenza virus RNA synthesis. Tat-SF1 interacted with free NP, but not with NP associated with RNA, and facilitated formation of RNA-NP complexes. These results suggest that Tat-SF1 may function as a molecular chaperone for NP, as does RAF-2p48/UAP56. This system has proven useful for further studies on the mechanism of influenza virus genome replication and transcription. molecular chaperone ͉ replication ͉ RNA-dependent RNA polymerase ͉ nucleoprotein

The heat shock response is an evolutionarily conserved, stress-responsive signaling pathway that adapts cellular proteostasis in response to pathologic insult. In metazoans, the heat shock response primarily functions through the posttranslational activation of heat shock factor 1 (HSF1), a stress-responsive transcription factor that induces the expression of cytosolic proteostasis factors including chaperones, cochaperones, and folding enzymes. HSF1 is a potentially attractive therapeutic target to ameliorate pathologic imbalances in cellular proteostasis associated with human disease, although the underlying impact of stress-independent HSF1 activation on cellular proteome composition remains to be defined. Here, we employ a highly controllable, ligand-regulated HSF1 that activates HSF1 to levels compatible with those that could be achieved using selective small molecule HSF1 activators. Using a combination of RNAseq and quantitative proteomics, we define the impact of stress-independent HSF1 activation on the composition of the cellular proteome. We show that stress-independent HSF1 activation selectively remodels cytosolic proteostasis pathways without globally influencing the composition of the cellular proteome. Furthermore, we show that stress-independent HSF1 activation decreases intracellular aggregation of a model polyglutamine-containing protein and reduces the cellular toxicity of environmental toxins like arsenite that disrupt cytosolic proteostasis. Collectively, our results reveal a proteome-level view of stress-independent HSF1 activation, providing a framework to establish therapeutic approaches to correct pathologic imbalances in cellular proteostasis through the selective targeting of HSF1.