A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

AbstractZoonotic coronavirus (CoV) infections, like those responsible for the current SARS-CoV-2 epidemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). While double-membrane vesicles (DMVs) appear to be a pan-coronavirus RO element, studies to date describe an assortment of additional coronavirus-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in coronavirus infection.

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Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes

mBio ◽

10.1128/mbio.00801-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 67

Author(s):

Helena J. Maier ◽

Philippa C. Hawes ◽

Eleanor M. Cottam ◽

Judith Mantell ◽

Paul Verkade ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Infectious Bronchitis Virus ◽

Rna Synthesis ◽

Rna Viruses ◽

Membrane Vesicles ◽

Viral Rna ◽

Double Membrane ◽

Infectious Bronchitis ◽

Positive Sense ◽

Infected Cells

ABSTRACTReplication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells ofex vivotracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCEAll positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

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The Transformation of Enterovirus Replication Structures: a Three-Dimensional Study of Single- and Double-Membrane Compartments

mBio ◽

10.1128/mbio.00166-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 86

Author(s):

Ronald W. A. L. Limpens ◽

Hilde M. van der Schaar ◽

Darshan Kumar ◽

Abraham J. Koster ◽

Eric J. Snijder ◽

...

Keyword(s):

Rna Synthesis ◽

Electron Tomography ◽

Three Dimensional ◽

Membrane Vesicles ◽

Viral Rna ◽

Membrane Structures ◽

Double Membrane ◽

Single Membrane ◽

Membrane Tubules ◽

Positive Strand Rna

ABSTRACTAll positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the familyPicornaviridae, have so far been described as either single- or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae. ET also revealed striking intermediate structures that strongly support the conversion of single-membrane tubules into double-membrane and multilamellar structures by a process of membrane apposition, enwrapping, and fusion. Collectively, our work unravels the sequential appearance of distinct enterovirus-induced replication structures, elucidates their detailed 3-D architecture, and provides the basis for a model for their transformation during the course of infection.IMPORTANCEPositive-strand RNA viruses hijack specific intracellular membranes and remodel them into special structures that support viral RNA synthesis. The ultrastructural characterization of these “replication structures” is key to understanding their precise role. Here, we resolved the three-dimensional architecture of enterovirus-induced membranous compartments and their transformation in time by applying electron tomography to cells infected with coxsackievirus B3 (CVB3). Our results show that closed single-membrane tubules are the predominant initial virus-induced structure, whereas double-membrane vesicles (DMVs) become increasingly abundant at the expense of these tubules as infection progresses. Additionally, more complex multilamellar structures appear late in infection. Based on compelling intermediate structures in our tomograms, we propose a model for transformation from the tubules to DMVs and multilamellar structures via enwrapping events. Our work provides an in-depth analysis of the development of an unsuspected variety of distinct replication structures during the course of CVB3 infection.

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RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Journal of Virology ◽

10.1128/jvi.76.8.3697-3708.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3697-3708 ◽

Cited By ~ 287

Author(s):

Rainer Gosert ◽

Amornrat Kanjanahaluethai ◽

Denise Egger ◽

Kurt Bienz ◽

Susan C. Baker

Keyword(s):

Electron Microscopy ◽

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Membrane Vesicles ◽

Rna Replication ◽

Viral Rna ◽

Double Membrane ◽

Ultrastructural Studies ◽

Infected Cells

ABSTRACT The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

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The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

mBio ◽

10.1128/mbio.00420-18 ◽

2018 ◽

Vol 9 (2) ◽

pp. e00420-18 ◽

Cited By ~ 11

Author(s):

C. E. Melia ◽

H. M. van der Schaar ◽

A. W. M. de Jong ◽

H. R. Lyoo ◽

E. J. Snijder ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Electron Tomography ◽

Membrane Vesicles ◽

Encephalomyocarditis Virus ◽

Escape Mutant ◽

Membrane Structures ◽

Double Membrane ◽

Single Membrane ◽

Phosphatidylinositol 4

ABSTRACTPicornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for theEnterovirusgenus of thePicornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of theCardiovirusgenus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCELike all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genusEnterovirus. The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

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The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Viruses ◽

10.3390/v11111030 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1030 ◽

Cited By ~ 10

Author(s):

Nicole Doyle ◽

Philippa C. Hawes ◽

Jennifer Simpson ◽

Lorin H. Adams ◽

Helena J. Maier

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Membrane Vesicles ◽

Viral Rna ◽

Double Membrane ◽

Severe Diarrhea ◽

The Usa ◽

History Of ◽

Host Cell Interactions ◽

Positive Strand Rna

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

10.1101/719443 ◽

2019 ◽

Author(s):

Nicole Doyle ◽

Philippa C. Hawes ◽

Jennifer Simpson ◽

Lorin H. Adams ◽

Helena J. Maier

Keyword(s):

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Membrane Vesicles ◽

Viral Rna ◽

Double Membrane ◽

Severe Diarrhea ◽

The Usa ◽

History Of ◽

Host Cell Interactions ◽

Positive Strand Rna

AbstractPorcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea and the virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to begin to address questions regarding virus-host cell interactions for this genera of coronavirus. Here we present a detailed study of PDCoV induced replication organelles. All positive strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta- and Gammacoronavirus genera have been characterized. However the structures induced by deltacoronaviruses, in particular the presence of convoluted membranes or double membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis and progeny particle release determined. Subsequently, virus induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double membrane vesicles. Significantly however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum and small associated tethered vesicles, double membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Journal of Virology ◽

10.1128/jvi.73.11.9555-9567.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9555-9567 ◽

Cited By ~ 148

Author(s):

Jason M. Mackenzie ◽

Malcolm K. Jones ◽

Edwin G. Westaway

Keyword(s):

Immunoelectron Microscopy ◽

Rna Synthesis ◽

Brefeldin A ◽

Specific Protein ◽

Viral Rna ◽

Membrane Structures ◽

Protein Marker ◽

Golgi Membranes ◽

Infected Cells ◽

Intermediate Compartment

ABSTRACT Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650–6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifyingtrans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.

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Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells

Journal of Virology ◽

10.1128/jvi.73.7.5957-5969.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 5957-5969 ◽

Cited By ~ 78

Author(s):

Stephanie T. Shi ◽

Jennifer J. Schiller ◽

Amornrat Kanjanahaluethai ◽

Susan C. Baker ◽

Jong-Won Oh ◽

...

Keyword(s):

Golgi Apparatus ◽

Rna Synthesis ◽

De Novo ◽

Hepatitis Virus ◽

Viral Proteins ◽

Viral Rna ◽

Membrane Structures ◽

Murine Hepatitis Virus ◽

Physical Evidence ◽

Infected Cells

ABSTRACT Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

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Studies of two temperature-sensitive mutants of Mengo virus

Canadian Journal of Biochemistry ◽

10.1139/o79-110 ◽

1979 ◽

Vol 57 (6) ◽

pp. 902-913 ◽

Cited By ~ 1

Author(s):

Patrick W. K. Lee ◽

John S. Colter

Keyword(s):

Rna Synthesis ◽

Viral Rna ◽

Temperature Sensitive ◽

Supporting Evidence ◽

Structural Polypeptide ◽

Infected Cells ◽

Mengo Virus ◽

In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

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

Journal of Virology ◽

10.1128/jvi.01917-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1339-1349 ◽

Cited By ~ 168

Author(s):

Tadasuke Naito ◽

Fumitaka Momose ◽

Atsushi Kawaguchi ◽

Kyosuke Nagata

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Nuclear Transport ◽

Intracellular Localization ◽

Viral Rna ◽

Polymerase Complex ◽

Virus Rna ◽

Binary Complexes ◽

Infected Cells ◽

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.

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