Localization of Influenza Virus Proteins to Nuclear Dot 10 Structures in Influenza Virus-Infected Cells

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Virology 310 (2003) 29Ð40 www.elsevier.com/locate/yviro

Localization of inﬂuenza virus proteins to nuclear dot 10 structures in inﬂuenza virus-infected cells

Yoshiko Sato,a Kenichi Yoshioka,a Chie Suzuki,a Satoshi Awashima,a Yasuhiro Hosaka,a Jonathan Yewdell,b and Kazumichi Kurodaa,c,*

a Department of Virology and Immunology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan b Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USA c Department of Immunology and Microbiology, Nihon University School of Medicine, Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan Received 22 July 2002; returned to author for revision 19 August 2002; accepted 4 December 2002

Abstract We studied influenza virus M1 protein by generating HeLa and MDCK cell lines that express M1 genetically fused to green fluorescent protein (GFP). GFP-M1 was incorporated into virions produced by influenza virus infected MDCK cells expressing the fusion protein indicating that the fusion protein is at least partially functional. Following infection of either HeLa or MDCK cells with influenza A virus (but not influenza B virus), GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in nuclear dots. Immunofluorescence revealed that the nuclear dots represent nuclear dot 10 (ND10) structures. The colocalization of authentic M1, as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence following in situ isolation of ND10. These findings demonstrate a previously unappreciated involvement of influenza virus with ND10, a structure involved in cellular responses to immune cytokines as well as the replication of a rapidly increasing list of viruses. © 2003 Elsevier Science (USA). All rights reserved.

Introduction controversial (Enami and Enami, 1996; Kretzschmar et al., 1996; Zhang and Lamb, 1996), it was shown that the cyto- Matrix protein (M1) consists of 252 residues and is the plasmic tails of HA and NA are essential for correct virus most abundant protein in influenza A virions, with about assembly (Mitnaul et al., 1996; Jin et al., 1997). The inter- 3000 molecules per spherical virion (Lamb, 1989). M1 action of M1 with lipids was shown in vitro by the efficient forms a continuous layer at the interior surface of the en- binding of purified M1 protein to liposomes (Bucher et al., velope (Nermut, 1972; Schulze, 1972; Booy et al., 1985; 1980; Gregoriades, 1980; Gregoriades and Frangione, 1981; Fujiyoshi et al., 1994) where it is believed to maintain virion Ruigrok et al., 2000). A minor component of influenza A integrity by bridging the viral envelope and the ribonucle- virion. NS2 [recently renamed nuclear export protein oprotein (RNP) complex. The interaction of M1 with both (NEP)] also associates with M1 (Yasuda et al., 1993; Ward viral integral membrane proteins in the plasma membrane et al., 1995). and newly assembled RNP complex is believed to be crucial M1 regulates RNP transport between cytoplasm and nu- in viral budding. M1 is thought to interact with the lipid cleus. At the penetration step, the exposure of influenza molecules of the envelope and also with the cytoplasmic virions to acidic pH in endosomes activates the proton tails of envelope proteins, hemagglutinin (HA), neuramini- channel activity of M2 protein, which acidifies the viral dase (NA), and M2. Although the latter interaction remains interior (Sugrue and Hay, 1991; Pinto et al., 1992). This induces the dissociation of M1 proteins from RNP (Zhirnov, 1990, 1992; Bui et al., 1996), which is essential for incom- * Corresponding author. Department of Immunology and Microbiol- ogy, Nihon University School of Medicine, Oyaguchi-kamicho, Itabashi- ing RNP to enter the nucleus (Martin and Helenius, 1991). ku, Tokyo 173-8610, Japan. Fax: ϩ81-3-3972-9560. At the late stage of infection, M1 binding to the newly E-mail address: [email protected] (K. Kuroda). formed RNP complexes in the nucleus induces the export of

RNP to the cytoplasm (Martin and Helenius, 1991; Bui et al., 2000). As the binding of M1 inhibits the transport of RNP into nuclei, the exported M1-RNP complex does not reenter the nucleus (Whittaker et al., 1996). NS2 (NEP) probably contributes to the export process (O’Neill et al., 1998). M1 binds RNA and inhibits the initiation of RNA synthesis in vitro (Hankins et al., 1990; Watanabe et al., 1996; Ye et al., 1989; Zvonarjev and Ghendon, 1980) and in vivo (Perez and Donis, 1998). The accumulation of M1 in the nucleus is postulated to mediate the suppression of mRNA synthesis by the polymerase complex of influenza virus late in infection (Shapiro et al., 1987; Zvonarjev and Ghendon, 1980). Other roles of M1 are suggested by its interaction with host proteins. By immunofluorescence, a portion of M1 colocalizes with actin filaments (Bucher et al., 1989), which may account for its resistance to detergent extraction (Ava- los et al., 1997). M1 also associates with histones (Zhirnov and Klenk, 1997) and the activated C-kinase (RACK 1), which may be involved in M1 phosphorylation (Reinhardt and Wolff, 2000). Phosphorylation is the only known post- Fig. 1. Expression of GFP/M1 in HeLa-GFP/M3 and MDCK-GFP/M10 translational modification of M1 (Gregoriades et al., 1984, cells. Panel A shows diffuse pattern of fluorescence from GFP/M1 in 1990). MDCK-GFP/M10 cells and also the image of phase-contrast microscopy in Despite its multiple roles in numerous cellular locales, the same field. (B) Polypeptides in MDCK-GFP (a), MDCK-GFP/M10 (b), the intracellular trafficking of M1 is largely uncharacter- HeLa-GFP (c), and HeLa-GFP/M3 (d) cells were separated by sodium ized. To study M1 in living cells, we constructed a green dodecyl sulfateÐpolyacrylamide gel electrophoresis and blotted to PVDF membranes. GFP/M1 protein on the membranes was detected by anti-PR8 fluorescent protein (GFP)/M1 fusion gene and established serum. The positions of molecular standards (kDa) are indicated at right. cell lines constitutively expressing the fusion protein. GFP from the jellyfish, Aequorea victoria, is a versatile tool for studying gene expression and trafficking of fusion protein in Immunoblot analysis confirmed that each of the lines vivo or in situ (reviewed in Lippincott-Schwartz et al., expressed a protein of the predicted molecular mass of the 1998; Chelbi-Alix et al., 1995; Tsien, 1998). A number of fusion protein (Fig. 1B). A protein of this mobility could viral proteins have been fused with GFP and their localiza- also be immunoprecipitated from detergent lysates of tion was traced in uninfected cells or cells infected with the MDCK-GFP/M10 cells labeled with [35S]methionine- source virus (Desai and Person, 1998; Elliott and O’Hare, [35S]cysteine (data not shown). Based on these results, we 1997; Presley et al., 1997; Thomas and Maule, 2000). Using concluded that GFP/M1 fusion protein is correctly ex- cells expressing GFP/M1 we found unexpectedly that M1 is pressed in each of the cell lines. localized to ND10 (nuclear dot 10), subnuclear structures that have been implicated in the replication of numerous Incorporation of GFP/M1 into virions viruses and in host cell responses to antiviral cytokines (reviewed in Hodges et al., 1998; Sternsdorf et al., 1997). To examine whether GFP/M1 fusion protein maintains the character of authentic M1 protein, we investigated the incorporation of GFP/M1 fusion protein into virions by Results sucrose density analysis. When the virus preparation from PR8-infected MDCK-GFP/M5-6 cells was fractionated by Expression of GFP/M1 protein in MDCK and HeLa cells velocity sucrose gradient (20 to 50%) centrifugation, we detected GFP/M1 by immunoblot analysis using anti-PR8 We established three cell lines expressing a GFP-M1 serum in the fractions 6 to 8 with the greatest amount in the fusion protein, MDCK-GFP/M10, MDCK-GFP/M5-6, and fraction 6, where the most HA protein was also detected HeLa-GFP/M3. In each of these lines the GFP fluorescence (Fig. 2A). The fractions 6 to 8 contained the bulk of virus was distributed uniformly throughout the cytoplasm and obtained from nontransfected MDCK cells and also from nucleus (Fig. 1A). This pattern of distribution was similar to MDCK-GFP cells (Fig. 2A) Western blotting analysis using that of M1 in previous reports where M1 was solely ex- anti-GFP antibody did not detect GFP protein in the frac- pressed (Bui et al., 1989, 1996). It should be noted, how- tions 6 to 8 of virions from GFP-MDCK cells even after ever, a patchy and reticular distribution of recombinant M1 10-fold concentration of virions in the fractions (Fig. 2B). was also reported (Zhao et al., 1998). The sucrose density analysis demonstrates that the GFP/M1 Y. Sato et al. / Virology 310 (2003) 29Ð40 31

GFP to M1 does not detectably alter its intracellular distribution, ability to associate with other viral structural proteins, or contribution to virus structure.

Inﬂuenza virus infection redistributes GFP/M1 to nuclear dots

We next examined the distribution of GFP/M1 in live PR8-infected MDCK-GFP/M5-6 cells (Fig. 3). We noted the first alteration in GFP/M1 distribution at ϳ4 h p.i. after which fluorescence began to concentrate in nuclear dots. By 10 h p.i. nearly all infected cells exhibited this pattern of fluorescence (Fig. 3H). Nuclear dots were distributed throughout nucleoplasm and were excluded from nucleoli (Fig. 3). Some cells continued to exhibit uniform fluorescence (Fig. 4A). Staining with anti-PR8 serum revealed that these cells were uninfected, and the accumulation of GFP/M1 in nuclear dots was only observed in infected cells (Fig. 4A and C). This demonstrates that the redistribution of GFP/M1 to nuclear dots is a direct result of virus infection and cannot be attributed to the effects of soluble cytokines released by infected cells. Similar findings were made with GFP/M1 expressing HeLa cells (data not shown). To confirm that the recruitment of GFP/M1 to nuclear dots reflects the properties of M1 and not GFP, we examined the effect of PR8 infection on HeLa-GFP and MDCK- GFP cells. The uniform diffuse pattern of fluorescence in MDCK-GFP and HeLa-GFP cells was not modified (Fig. 4B and D), even following 24-h infection (data not shown). These results indicate that the M1 moiety of GFP/M1 fusion protein is required for localization of GFP/M1 to nuclear dots.

Fig. 2. Sucrose gradient centrifugation analysis of virions grown in Influenza B virus does not induce punctate accumulation MDCK-GFP/M5-6 cells. Sucrose gradient centrifugation analysis of viri- of GFP/M1 ons grown in MDCK-GFP/M5-6, MDCK-GFP, and MDCK cells were infected with PR8 for 15 h and virions were prepared from the supernatants To characterize the requirements for movement of of these cells. The virions were overlaid onto linear 20Ð50% sucrose gradients and centrifuged. The gradients were fractionated from the bot- GFP/M1 to nuclear dots, we examined the effects of other tom. (A) Polypeptides in each fraction were analyzed by immunoblot influenza viruses on GFP/M1 distribution. MDCK-GFP/ analysis using anti-PR8 serum. PR8-infected cells (IC) were also analyzed. M5-6 cells were infected with A/Aichi/2/68, which be- (B) Virions in fractions 6, 7, and 8 were pelleted to concentrate and longs to H3 influenza A virus subtype or with influenza B solubilized by sodium dodecyl sulfateÐpolyacrylamide gel electrophoresis sample buffer. The solubilized virions were analyzed by immunoblot virus, B/Yamagata/1/73. The amino acid sequence of M1 analysis using anti-GFP monoclonal antibody. PR8-infected MDCK, protein of PR8 shows different homology with that of MDCK-GFP, and MDCK-GFP/M5-6 cells were also analyzed. these viruses, 96.8% and 29.0% homology with A/Aichi/ 2/68 and B/Yamagata/1/73, respectively. Ten h p.i., nu- is specifically incorporated into influenza virus particles and clear dots of GFP/M1 were clearly observed in A/Aichi/ its incorporation does not grossly modify virus sedimenta- 2/68-infected cells, whereas no punctate pattern was in tion. the cells infected with B/Yamagata/1/73 (Fig. 5) (or cells Incorporation was also suggested by the observation of infected with B/Bangkok/163/90). Notably, the nuclear fluorescent particles on erythrocytes when erythrocytes dots induced by A/Aichi/2/68 were smaller and more were incubated with the supernatants of PR8-infected numerous than those induced by PR8. Smaller nuclear MDCK-GFP/M5-6 cells (data not shown). dots were observed cells infected with A/Udorn/307/72, We conclude from these data that the genetic fusion of another H3 subtype virus. 32 Y. Sato et al. / Virology 310 (2003) 29Ð40

Fig. 3. Time course of nuclear punctate dot induction of GFP/M1 in PR8 infected cells. MDCK-GFP/M5-6 cells were adsorbed with PR8 of moi 10 at 4¡C and then incubated at 37¡C. Images of phase-contrast (A, C, E, and G) and conventional ﬂuorescence (B, D, F, and H) microscopy of the infected cells were taken in the almost same ﬁeld at every 1-h incubation. No obvious change was observed until 4-h incubation, when accumulation of GFP/M1 in nuclear dots began to appear. Images acquired 4, 6, 8 and 10 h p.i. are shown. Arrowheads identify identical cell for points of reference. moi, multiplicity of infection.

Identification of GFP/M1 containing nuclear dots greater number detected by anti-SC35 antibody. As reported previously (Guldner et al., 1992; Fortes et al., 1995; Wolff Influenza virus infection is known to alter the architec- et al., 1998), PR8 infection increased the number and de- ture of nuclear substructures, including PML-containing creased the size of PML- and SC35-containing dots (data ND10 (Guldner et al., 1992) and SC35-containing speckled not shown). The expression of GFP/M1 in HeLa cells did domains (Fortes et al., 1995). We examined the relationship not affect the staining pattern with anti-SC35 and anti-PML between GFP/M1 containing nuclear dots and these struc- antibodies. tures using antibodies specific for PML or SC35. Staining of Following PR8 infection, GFP/M1 containing dots sel- HeLa cells with each antibody revealed nuclear dots, with a dom coincided staining by anti-SC35 antibodies (Fig. 6A, Y. Sato et al. / Virology 310 (2003) 29Ð40 33

Fig. 4. Punctate accumulation of GFP/M1 appeared only in viral antigen-positive cells. MDCK-GFP/M10 (A and C) and MDCK-GFP (B and D) cells were infected with PR8 at moi 10. The cells were fixed and permeabilized at 10 h p.i., and then stained with anti-PR8 serum followed by rhodamine-conjugated secondary antibody. The images of conventional fluorescence microscopy of GFP/M1 (A), GFP (B), and rhodamine (C, D) are shown. Arrows identify an uninfected cell. Arrowhead identifies an cell not expressing the GFP/M1 fusion protein. moi, multiplicity of infection; GFP, green fluorescent protein.

C, and E). Even in the infrequent coinciding dots, the size of Colocalization of NS1 and NS2 proteins with M1 protein GFP/M1 dot and that of coinciding speckled domains were in ND10 grossly different. In contrast, the nuclear dots of GFP/M1 were often almost completely superimposable with dots The infection-dependent localization of GFP/M1 to stained with anti-PML antibody at least until 10 to 15 h p.i. ND10 suggests that other viral proteins are involved in this (Fig. 6B, D, and F). process. As NS2 protein is known to associate with M1 We next examined whether M1 produced by influenza protein (Yasuda et al., 1993; Ward et al., 1995) and NS1 virus localizes to ND10. Although M1 protein in influenza induces the redistribution of nuclear substructure (Fortes et virus-infected cells distributes uniformly throughout cyto- al., 1995; Wolff et al., 1998), these two proteins are the plasm and nucleus (Fig. 7C) it is possible that the excess of prime candidates for participating in M1 localization to M1 in the nucleus obscures the M1 localized in nuclear dots. ND10. We therefore examined the distribution of NS1 and/or NS2 with M1 following the four-step extraction To test this idea, PR8-infected MDCK or HeLa cells at- procedure using anti-NS1 or anti-NS2 antibodies. This re- tached to coverslips were subjected to a four-step extraction vealed that both NS1 and NS2 were present in M1-contain- procedure originally developed to isolate the nuclear matrix, ing ND10 (Fig. 9). This observation suggests the involve- but which also isolates components of nuclear dots (Anton ment of NS1 and/or NS2 in the localization of M1 to ND10. et al., 1999; Staufenbiel and Deppert, 1984). Indeed, M1 was localized to nuclear dots following this procedure, as was GFP/M1 (Fig. 7A, B, C, and D). The staining of Discussion PR8-infected HeLa cells with anti-PML antibody after the extraction revealed the colocalization of M1 with ND10 In the present report we demonstrate that it is possible to (Fig. 8). label influenza virus M1 with GFP and maintain M1 func- It should be noted that the staining intensity with anti- tion as demonstrated most convincingly by incorporation of PML antibody was different between M1 antigen-positive M1/GFP into virions. These encouraging results have and negative cells. The dots of PML were much clearer in prompted our attempts to generate influenza virus geneti- M1 antigen-negative cells, which might reflect the previous cally engineered to express GFP/M1, which would be a observation that the size and number of ND10 is altered in valuable tool with numerous applications, although we have influenza virus-infected cells (Guldner et al., 1992). not yet succeeded. Based on these results, we concluded that a fraction of Unexpectedly, we found that GFP/M1 localizes to ND10 authentic M1 protein localize in ND10. following infection with influenza A viruses. On the other 34 Y. Sato et al. / Virology 310 (2003) 29Ð40

Fig. 5. A/Aichi/2/68 but not B/Yamagata/1/73 induces the accumulation of GFP/M1 in nuclear dots. HeLa-GFP/M5-6 cells were infected with A/Aichi/2/68 (A, B, and C) or B/Yamagata/1/73 (D, E, and F) at moi 10 for 10 h. The infected cells were fixed and permeabilized, and then stained with anti-H3 or anti-B serum followed by rhodamine-conjugated secondary antibody. The images of phase-contrast microscopy (A and D) and those of conventional fluorescence microscopy of rhodamine (C and F) and GFP/M1 (B and E) are shown. A, B, and C, and D, E, and C were taken in the same field respectively. moi, multiplicity of infection. hand, the infection with influenza B viruses does not induce may, however, reflect temporal differences in expression the relocalization of GFP/M1. Influenza B virus is highly and not differences in structure/function. The targeting of similar to influenza A virus in its replication strategy and M1/GFP to ND10 may be favored by virtue of the fact that effects on infected cells. Natural stable reassortants between it is presynthesized by cells. In this scenario, the association influenza A and B viruses, however, have never been ob- of newly synthesized M1 with viral proteins would disfavor served (Ghenkina and Ghendon, 1979; Lamb and Krug, its transport to ND10. It is also possible that the attachment 1996) despite that both types infect humans. The most likely of GFP increases the propensity of M1 to localize to ND10 reason of this phenomenon is the inability of influenza A by either direct (e.g., binding to a ND10 protein) or indirect virus proteins to interact functionally with influenza B virus (e.g., increasing association with a protein that binds to proteins, as shown in the case of RNP protein components ND10) mechanism. Even if this is correct, we note that the (Crescenzo-Chaigne et al., 1999; Jambrina et al., 1997). fusion protein exaggerates the normal propensity of M1 to Thus, these findings strongly suggest that the virus-induced localize to ND10. redistribution of GFP/M1 is based on the interaction of The colocalization of both of NS1 and NS2 to M1- GFP/M1 with viral proteins and not general alterations in containing ND10 suggests the involvement of these proteins the cell that accompany viral infection. in the localization of M1 and M1-GFP to ND10. Further Although GFP/M1 should maintain important elements experiments are required to determine whether these pro- of native structure, as indicated by its incorporation into teins alone, or in conjunction are able to induce M1 local- virions, it appears to differ from authentic M1, which unlike ization to ND10. It will be of interest to determine to what M1/GFP less strongly localizes to ND10. This difference extent these proteins possess intrinsic ND10 localization Y. Sato et al. / Virology 310 (2003) 29Ð40 35 signals. If, for example, NS2 possesses such a signal, a Materials and methods relatively simple scenario would be that M1 is recruited to ND10 by virtue of its binding to NS2. Cell cultures, viruses, and virus infection ND10 is a nuclear substructure originally discovered as targets of autoantibodies in patients suffering from the au- Influenza viruses, A/PR/8/34 (PR8), A/Aichi/2/68, toimmune disease primary biliary cirrhosis (Sternsdorf et A/udorn/307/72, B/Yamagata/1/73, and B/Bangkok/163/90 al., 1997). A number of proteins are known to be associated were grown in the allantoic cavities of 10-day-old embry- with ND10 including PML, SP100, SUMO-1 (PIC-1), onated chicken eggs and allantoic fluids were used as virus NDP-55, NDP-52, and int-6 (reviewed in Hodges et al., stocks. Virus stocks were aliquoted and stored at Ϫ80¡C. 1998). Of these proteins, PML is best characterized and is MDCK and HeLa cells were passaged in Eagle’s mini- believed to be involved in negative growth regulation and mum essential medium (MEM) containing 10% fetal calf apoptosis (Wang et al., 1998b; Torii et al., 1999; Quignon et serum (FCS). MDCK-GFP/M10, MDCK-GFP/M 5-6, al., 1998; Mu et al., 1994; Koken et al., 1995; Liu et al., HeLa-GFP/M3, MDCK-GFP, and HeLa-GFP cells were 1995; Le et al., 1996). Mice with a targeted deletion of this passaged in MEM containing 10% FCS and 1 mg/ml G 418 gene are more susceptible to tumorigenesis and infections (Nacalai Tesque). (Wang et al., 1998a). ND10 is a target of a rapidly growing Cells washed with phosphate-buffered saline (PBS) were list of viruses. The function of ND10 in viral replication is infected at 4 or 37¡C with influenza viruses in MEM con- obscure, but it has been proposed to be involved in coordi- taining 1% bovine serum albumin (BSA) at moi (multiplic- nating the expression and replication of viral genomes. A ity of infection) 10 for 30 min at 37¡Cand1hat4¡C. At the number of DNA viruses, such as SV40, adenovirus 5, and end of adsorption, cells were washed with PBS and incu- HSV-1 begin replication in the periphery of ND10 (re- bated further in MEM at 37¡C. viewed in Sternsdorf et al., 1997; Maul, 1998). Viral proteins like tax of HTLV-1, E4-ORF3 of adenovirus 5, IE72 Antibodies (IE1) of HCMV, and ICP0 (Vmw110) of HSV-1 have been reported to interact with ND10 and alter its structure (Des- Monoclonal antibodies anti-PML (PG-M3) and anti- bois et al., 1996; Everett and Maul, 1994; Doucas et al., SC35 (␣SC35) were purchased from Santa Cruz Biotech- 1996; Korioth et al., 1996). The degradation of PML by nology and BD PharMingen, respectively. PG-M3 is human herpesvirus was also reported (Chelbi-Alix and de The, PML-specific antibody and does not bind to PML in MDCK 1999). cells. Anti-GFP monoclonal antibodies were purchased It was previously reported that influenza virus infection from Clontech (JL-8). For anti-M1antibodies, rabbit serum alters the size and the number of ND10 (Guldner et al., raised against recombinant M1 protein, which was kindly 1992), a finding we confirmed. The expression of PML is provided by Dr. M. Enami (Kanazawa University School of induced by interferons (Chelbi-Alix et al., 1995; Lavau et Medicine, Japan), and monoclonal antibody M2/1C6 were al., 1995; Stadler et al., 1995) and overexpression of PML used. Rabbit serum raised against NP protein of PR8, re- suppresses the replication of several viruses including in- combinant NS1 protein, and recombinant NS2 were gener- fluenza virus (Chelbi-Alix et al., 1998). These results sug- ous gifts of Dr. S. Tamura (National Institute of Infectious gest an important role of PML in the interferon-induced Diseases, Japan), Dr. M. Enami, and Dr. A. Ishihama (Na- antivirus state. tional Institute of Genetics, Japan), respectively. Rabbit sera We propose that the accumulation of M1, NS1, and NS2 against whole viruses, anti-A/udorn/307/72 (kindly pro- in ND10 of influenza virus-infected cells induces the dys- vided by Dr. K. Shimizu, Nihon University School of Med- function of ND10 or proteins associated with ND10 like icine, Japan), anti-B/Yamagata/1/73 (kindly provided by PML, thereby interfering with the interferon-induced anti- Dr. T. Odagiri, National Institute of Infectious Diseases, viral state, including the induction of apoptosis. In this Japan), and anti-PR8 were also used. Anti-A/udorn/72 and scenario, influenza virus would utilize a supplementary anti- anti-B/Yamagata/1/73 sera were used as anti-H3 subtype interferon mechanism in addition to that mediated by NS1 and anti-B type antibodies, respectively. (Garcia-Sastre et al., 1998; Talon et al., 2000; Bergmann et al., 2000). Another possible role of PML in virus infection Construction of pGFP/M1 is suggested by a recent report that showed PML controls expression of genes crucial for MHC class l-restricted an- Plasmid pGFP/M1, expression vector for GFP/M1 fusion tigen presentation (Zheng et al., 1998). Influenza virus protein, was constructed as follows. A DNA fragment con- might interfere with the immune response of hosts by dis- taining coding region of M1 gene was amplified from pT3/ rupting the function PML or ND10. PR8-M (kindly provided by Dr. M. Enami) by polymerase In summary, our data strongly suggest a novel interaction chain reaction using two primers, M-F (AGATCTAT- of M1, NS1, and NS2 with ND10, an important nuclear GAGTCTTCTAACCGAG) and M-R (TCCCCCGGGT- structure. Further elucidation of this interaction should shed CACTTGAACCGTTGCAT). The amplified DNA frag- new light on the role of ND10 in influenza virus infections. ment and pEGFP-C1, a GFP fusion protein expression 36 Y. Sato et al. / Virology 310 (2003) 29Ð40

Fig. 6. Nuclear dots of GFP/M1 coincide with PML-containing ND10. HeLa- GFP/M3 cells were infected with PR8 at moi 10 for 15 h, fixed and permeabilized, and then stained with anti-SC35 or anti-PML monoclonal antibody followed by rhodamine-conjugated secondary antibody. The fluorescence of GFP/M1 (C and D) and rhodamine (A and B) of the stained cells were observed simultaneously by laser confocal microscopy. A and C are paired micrographs of anti-SC35 antibody stained cells. B and D are paired micrographs of anti-PML antibody stained cells. E and F are merged images of A and C, and B and D, respectively. moi, multiplicity of infection. Fig. 8. Nuclear dots of authentic M1 also colocalize with ND10. HeLa cells were infected with PR8 at moi 10 for 6 h and then in situ extracted. The residual structures were fixed and stained with anti-M1 rabbit serum and anti-PML mouse monoclonal antibody followed by rhodamine-conjugated anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibodies. The stained cells were observed with conventional fluorescence microscope for rhodamine (A) and FITC (B). The black and white pictures obtained by CCD-camera were false colored (red, rhodamine; green, FITC) and merged (C). moi, multiplicity of infection; ND10, nuclear dot 10. Y. Sato et al. / Virology 310 (2003) 29Ð40 37

Fig. 9. Colocalization of NS1 and NS2 proteins with M1 protein in extracted cells. MDCK cells were infected with PR8 at moi 10 for 12 h and were extracted in situ by a four-step procedure and the residual structures were fixed. The structures were stained with anti-M1 monoclonal antibody and anti-NS1 (A, B, and C) or anti-NS2 serum (D, E, and F) followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and rhodamine-conjugated anti-rabbit IgG antibodies. The stained cells were observed with conventional fluorescence microscope for FITC (A and D) and rhodamine (B and E). The black and white pictures obtained by CCD-camera were false colored (green, FITC; red, rhodamine) and merged (C and F). vector obtained from Clontech, were double-digested with Immunoblot analysis BglII and SmaI. The digested fragment and the vector were ligated to obtain pGFP/M1 in which GFP gene was attached Polypeptides in samples were separated under reducing to the N-terminus of M1 protein. The fusion protein ex- conditions by sodium dodecyl sulfate-polyacrylamide gel pressed from pGFP/M1 should have an additional 5 amino electrophoresis (SDS-PAGE). The separated polypeptides acids (Ser-Gly-Leu-Arg-Ser) derived from polylinker re- were electrotransferred onto PVDF membranes (Pall) by the gion of pEGFP-C1 between asparagine of GFP C-terminus semidry method. The membranes were then incubated in and methionine of M1 N-terminus. blocking buffer [TBS-T (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20) containing 10% skim milk] for 1 h and washed with TBS-T. They were subsequently in- Establishment of cell lines expressing GFP/M1 fusion cubated with rabbit sera or monoclonal antibodies for 1 h protein and washed with TBS-T. Finally, secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG anti- Plasmid pGFP/M1 was introduced into MDCK and bodies (Amersham Pharmacia Biotech) were applied to the HeLa cells with liposome-mediated transfection using membranes. After washing with TBS-T, the peroxidase- DOTAP (Boehringer Mannheim). The transfected cells conjugated antibodies on the membranes were detected by were cultured in MEM containing 10% FCS and 2 mg/ml using the ECL system (Amersham Pharmacia Biotech) ac- G418 to select the cells that express GFP/M1 fusion protein cording to the manufacturer’s manual. The images of the constitutively. The survived cells were cloned by limiting chemiluminescence were captured with a CCD-camera dilution method or colony isolation, and each clone was (LAS-1000, Fujifilm). tested for the expression of GFP/M1 by fluorescence microscopy and flow cytometry using FACScan (Becton Dick- inson). MDCK-GFP/M10, MDCK-GFP/M5-6, and HeLa- Sucrose gradient centrifugation analysis of virus GFP/M3 were chosen because of their moderate intensity of fluorescence than others. As a control plasmid, pEGFP-C1 The supernatants of influenza virus infected cells were was also introduced to MDCK and HeLa cells and cell lines clarified by low-speed centrifugation and then the virions MDCK-GFP and HeLa-GFP, which express authentic GFP, were pelleted by the centrifugation of 200,000 ϫ g for2h were established by the same way as above. at 4¡C. The pelleted virions were resuspended in NTE 38 Y. Sato et al. / Virology 310 (2003) 29Ð40

Fig. 7. Nuclear dots of authentic M1 protein in extracted cells. MDCK-GFP/M10 (A and B) and MDCK (C and D) cells were infected with PR8 at moi 10 for 10 h. The infected MDCK-GFP/M10 cells were extracted in situ by a four-step procedure and the residual structures were fixed and observed by phase-contrast (A) and conventional fluorescence microscopy (B) in the same field. The infected MDCK cells were fixed before (C) or after (D) in situ extraction, and then stained with anti-M1 monoclonal antibody (C and D) followed by rhodamine-conjugated anti mouse IgG (C and D). The stained cells were observed with conventional fluorescence microscope for rhodamine (C and D). moi, multiplicity of infection. buffer [10 mM Tris (pH 7.4), 100 mM Na Cl, 1 mM EDTA] Indirect immunofluorescence microscopy and applied on a linear 20Ð50% sucrose gradient. The fractionation of virions by velocity sucrose gradient centrif- Cells were grown on glass coverslips in MEM con- ugation was done in a RPS56T (Hitachi) rotor (35,000 rpm, taining 10% FCS. Where indicated, cells were infected 1h,4¡C). The gradients were fractionated from the bottom with influenza viruses for the indicated times. After into 250-␮l samples. For the immunoblot analysis, 5 ␮lof washing with PBS, cells were fixed for 10 min in PBS each fraction was solubilized with SDS-PAGE sample containing 4% paraformaldehyde, permeabilized for 10 buffer and boiled for 5 min and applied to 11% polyacryl- min in PBS containing 0.2% Triton X-100, and incubated amide-SDS gels. To analyze concentrated virions in the for 30 min in PBS containing 3% BSA (3% BSA-PBS). fractions, 50 ␮l of each fraction was centrifuged at 40,000 Subsequently, cells were stained for1hwithprimary rpm for 90 min using an RP80 AT rotor (Hitachi) and antibodies or sera diluted in 3% BSA-PBS. The cells SDS-PAGE sample buffer was added to the pelleted virions. were washed and incubated with Alexa 350-, rhodamine, The solubilized virions were analyzed by immunoblot tech- or fluorescein isothiocyanate (FITC)-conjugated goat an- nique. ti-rabbit immunoglobulin G (IgG) (Molecular Probe or Cappel), and/or Alexa 350-, rhodamine-, or FITC-conjugated goat anti-mouse IgG (Molecular Probe or Cappel) In situ cell extraction diluted in 3% BSA-PBS. Finally, the coverslips were washed with PBS and mounted in Fluoromount-G Cells cultured on glass coverslips were extracted by the (Southern Biotechnology Associates). If it is not stated, method of Staufenbiel and Deppert (1984). Briefly, cells cells were viewed on an Olympus IX70 conventional were washed with KM buffer [10 mM N-morpholinoeth- fluorescence microscope for immunofluorescence analy- anesulfonic acid (pH 6.2), 10 mM NaCl, 1.5 mM MgCl , 2 sis, and images were photographed with PROVIA (Fuji- 10% glycerol, 10 ␮g/ml aprotinin] and incubated on ice in film) or captured with a digital camera (PDMC Ie/OL, KM buffer containing 1% NP40, 1 mM EGTA, 5 mM Olympus or Orca-ER, Hamamatsu Photonics). For con- dithiothreitol (first extraction step). After wash with KM focal laser scanning microscopy, a Zeiss LSM410 invert buffer, structures on coverslips were incubated at 37¡Cin ϫ KM buffer containing 50 ␮g/ml DNase I (second extraction microscope equipped with 63 objective lens was used. step). The DNase I solution was removed and KM buffer containing 2 M NaCl, 1 mM EGTA, and 5 dithiothreitol was added and incubated at 4¡C (third extraction step). Then the Acknowledgments structures were washed with KM buffer and incubated at 37¡C in KM buffer containing 50 ␮g/ml DNase I and 50 We acknowledge Dr. Masaki Imai and Dr. Tatsuya Sakai ␮g/ml RNase A (fourth extraction step). for assistance with confocal laser scanning microscopy. Y. Sato et al. / Virology 310 (2003) 29Ð40 39

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