Fumitaka Momose

Involvement of Hsp90 in Assembly and Nuclear Import of Influenza Virus RNA Polymerase Subunits

ABSTRACT 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.

Cellular Splicing Factor RAF-2p48/NPI-5/BAT1/UAP56 Interacts with the Influenza Virus Nucleoprotein and Enhances Viral RNA Synthesis

ABSTRACT 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.

Apical Transport of Influenza A Virus Ribonucleoprotein Requires Rab11-positive Recycling Endosome

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.

Replication-Coupled and Host Factor-Mediated Encapsidation of the Influenza Virus Genome by Viral Nucleoprotein

ABSTRACT 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.

Identification of host factors that regulate the influenza virus RNA polymerase activity

Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.

Influenza A virus hemagglutinin and neuraminidase mutually accelerate their apical targeting through clustering of lipid rafts.

ABSTRACT 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. IMPORTANCE Lipid 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.

Involvement of vesicular trafficking system in membrane targeting of the progeny influenza virus genome

The genome of influenza type A virus consists of single-stranded RNAs of negative polarity. Progeny viral RNA (vRNA) replicated in the nucleus is nuclear-exported, and finally transported to the budding site beneath the plasma membrane. However, the precise process of the membrane targeting of vRNA is unclear, although viral proteins and cytoskeleton are thought to play roles. Here, we have visualized the translocation process of progeny vRNA using fluorescence in situ hybridization method. Our results provide an evidence of the involvement of vesicular trafficking in membrane targeting of progeny vRNA independent of that of viral membrane proteins.

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release : relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C‐termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag‐to‐GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag‐to‐GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag‐to‐GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.

Defect of Human Immunodeficiency Virus Type 2 Gag Assembly in Saccharomyces cerevisiae

ABSTRACT We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.