Association of Influenza Virus Matrix Protein with Ribonucleoproteins May Control Viral Growth and Morphology

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Virology 304, 89–96 (2002) doi:10.1006/viro.2002.1669

Teresa Liu,* Jacqueline Muller,† and Zhiping Ye*,1

*Laboratory of Pediatric and Respiratory Viral Diseases, †Laboratory of Vector-Borne Viral Disease, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics and Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received April 25, 2002; returned to author for revision June 25, 2002; accepted July 12, 2002

The matrix protein (M1) of influenza virus plays a central role in viral replication. In relation to viral growth and morphology, we studied the RNP-binding activity of M1s from high-growth strain A/Puerto Rico/8/34 (A/PR8/34) and the relatively low-growth wild-type strain A/Nanchang/933/95. The RNP-binding strength of M1 was studied by disruption of M1 from M1/RNP complexes with salt and acidic condition. Our results indicated that binding of M1 of high-growth A/PR8/34 was more difficult to break than the binding of M1 of low-growth A/Nanchang/933/95. Consistent with the presence of M1 in A/PR8/34, binding of M1 of Resvir-9, a reassortant containing P, M, and NS genes from A/PR8/34 and the rest of genes from A/Nanchang/933/95 and retaining relative high-growth characteristic, was relatively difficult to break than the binding of M1 of A/Nanchang/933/95. Physical properties of morphological features of these viruses were studied by velocity sucrose gradient centrifugation and transmission electron microscopy of purified viral particles, and by immunofluorescence staining of hemagglutinin expressed on the surface of infected cells. The results demonstrated that high-growth strains, A/PR8/34, and a relative high-growth reassortant, Resvir-9, had characteristics associated predominantly with spherical particles, while the low-growth strain, A/Nanchang/933/95, had characteristics of filamentous particles. These studies indicate that the binding between M1 and RNP complex might determine viral growth and morphology. © 2002 Elsevier Science (USA)

INTRODUCTION gered by transport of Hϩ ions across the viral membrane by M2 (Sugrue and Hay, 1991; Helenius, 1992; Lamb et The genome of influenza A virus consists of eight al., 1994). Later in the replication cycle, newly synthe- distinct segments of negative-sense RNA coding for at sized M1 migrates to the nucleus of the influenza virus- least 10 viral proteins of which three are known to func- infected cell where it again comes into close proximity tion as polymerases. The viral RNA, nucleoprotein (NP), with newly synthesized RNP (Bui et al., 1996; Patterson et and polymerases are in close association in the ribonu- al., 1988). The binding of M1 with RNP leads to the cleoprotein (RNP) (Heggeness et al., 1982; Lamb and transport of M1/RNP complexes from the nucleus to the Choppin, 1983; Murti et al., 1988). Matrix protein (M1) is cytoplasm (Martin and Helenius, 1991; Huang et al., located between the RNP and the inner surface of the 2001), and to prevent RNP from reentering the nucleus lipid envelope in the intact virion (Allen et al., 1980; (Martin and Helenius, 1991). The interaction of M1 with Bucher et al., 1980; Schulze, 1972). Two major external HA, NA, M2, and lipid membranes also indicates a role glycoproteins, hemagglutinin (HA) and neuraminidase for M1 in the budding of virions from the cell surface (NA), and a small protein M2, serving as a transmem- (Bucher et al., 1980; Gregoriades, 1980; Robertson et al., brane channel, are anchored in the viral envelope (Lamb 1982; Ye et al., 1987; Enami and Enami, 1996; Lamb et al., et al., 1985; Sugrue and Hay, 1991). M1, as a multifunctional protein, is not only the major 1985; Lamb and Choppin, 1983). In the maturation of viral structural component of the virion, but also plays an particles, the M1:NP ratio of viral particles influences important role in many steps in the replication of the morphological features and infectivity of the released influenza virus. During early viral replication, dissociation viruses (Roberts et al., 1998). of M1 from RNP is required for the entry of viral RNP into The interactions of M1 with RNP have been studied the cytoplasm of the host cell (Bui et al., 1996; Helenius, extensively (Baudin et al., 2001; Compans and Klenk, 1992; Martin and Helenius, 1991). Dissociation is trig- 1979; Rees and Dimmock, 1982; Ruigrok and Baudin, 1995; Schulze, 1972; Ye et al., 1999). The amino acid sequences of M1 needed for binding to RNP have been

1 studied using RNP isolated from virions and M1 synthe- To whom correspondence and reprint requests should be ad- sized in vitro (Ye et al., 1987, 1999). The binding domains dressed at Center for Biologics and Evaluation and Research, Food and Drug Administration, Building 29A, Room 2B17, 8800 Rockville Pike, of M1 protein to RNP has been studied by examining the Bethesda, MD 20982. Fax: (301) 840-3157. E-mail: [email protected]. association of purified RNPs with 35S-labeled M1 trans-

TABLE 1 tive studies of viral infectivity titers in MDCK cell by Growth Characteristics of A/PR8/34, A/Nanchang/933/95, measurement of plaque-forming units (PFU). As summa- and Resvir-9 Viruses rized in Table 1, A/PR8/34 had approximately 50-fold greater plaque yield (9.8 ϫ 10 7 PFU/ml) than A/Nan- a Virus PFU/ml HAU/ml PFU/HAU chang/933/95 (2 ϫ 10 6 PFU/ml). The reassortant, Res- vir-9, had an intermediate yield (1.2 ϫ 10 7 PFU/ml), which A/PR/8/34 9.8 ϫ 107 2048 4.8 ϫ 104 Resvir-9 1.3 ϫ 107 512 2.3 ϫ 104 yielded viral infectivity titer sixfold greater than that of A/Nanchang/933/95 2.0 ϫ 106 256 4.0 ϫ 103 low-growth A/Nanchang/933/95. Table 1 also shows the titer of HA from virus after 48 h replication in eggs. a The plaque-forming units were determined by plaque assay in A/PR8/34 had the highest HA titer (2048 HAU), while ␮ MDCK cells in the presence of trypsin (2 g/ml). The data were means A/Nanchang/933/95 had the lowest HA titer (256 HAU). of three independent experiments, and the standard deviation of each experimental point was below 10%. Resvir-9 had an intermediate HA titer (512 HAU). The genotype of the reassortant was determined by polymerase chain reaction-restriction fragment length polymor- lated from cDNAs coding for either the wild-type phism (PCR-RFLP) (Offringa et al., 2000). As shown in A/WSN/33 M1 or the substitution and deletion mutants of Table 2, Resvir-9 received its NA, HA, and NP genes from WSN M1. The effects of reconstituting these wild-type A/Nanchang/933/95 but the rest of the internal genes and mutant M1s with RNP under conditions of altered including M was from A/PR8/34. The fact that Resvir-9 pH, ionic strength, and added detergent are used to did not replicate as efficiently as A/PR8/34 suggests that further define the interacting domains of M1. With RNP NA, HA, and NP genes from A/PR8/34 also contribute to isolated from virions and M1 synthesized in vitro,M1 high-growth characteristics. binds to RNP not only by specific binding sites for viral RNA (vRNA) but also by what appears to be direct inter- A/PR8/34, A/Nanchang/933/95, and Resvir-9 viruses action of M1 with NP (Ye et al., 1999). differ in morphology The biological significance of M1 protein binding to It has been reported that filamentous viruses may RNP has not been studied extensively. It has been re- replicate to lower titer and that conversion to spherical ported that at acidic conditions in which M1 protein morphology may confer higher growth properties (Burnet dissociated from RNP resulted in reduction of transpor- and Lind, 1957; Kilbourne and Murphy, 1960). The long tation of RNP from nucleus to cytosol (Bui et al., 1996). Our recent studies demonstrate that vRNA and M1 must both be present in order for NP to assemble into the TABLE 2 quaternary helical structure of viral RNP (Huang et al., 2001). In this article, we compared the RNP-binding ac- Genotyping of A/PR8/34 (PR8), A/Nanchang/933/95 (Nan), and Resvir-9 Viruses tivity of the M1 proteins from A/PR8/34 virus (high- growth), A/Nanchang/933/95 (relatively low-growth), and Gene segment PR8 Resvir-9 Nanchang a reassortant between A/PR8/34 and A/Nanchang/ 933/95 and studied the relationship of M1-RNP binding 1 PB1 PR8 PR8 Nan to replication and morphogenesis of influenza viruses, 2 PB2 PR8 PR8 Nan suggesting that association of influenza virus matrix pro- 3 PA PR8 PR8 Nan 4 HA PR8 Nan Nan tein with RNP controls viral growth and morphology. 5 NP PR8 Nan Nan 6 NA PR8 Nan Nan RESULTS 7 M PR8 PR8 Nan 8 NS PR8 PR8 Nan Generation and characterization of high-growth reassortant from high-growth A/PR8/34 and Note. The genotype of the reassortant was determined by reverse low-growth A/Nanchang/933/95 transcription-polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism (RFLP) (Offringa et al., 2000). Briefly, cDNAs Although the critical role of interaction between M1 of the viruses were synthesized by reverse transcription using the Ј and RNP in functional and morphological features of universal primer complementary to the conserved 3 end of the genomic RNA. Subsequent amplification of the resulting cDNAs (ex- influenza viral replication is apparent, the mechanism of cept for the larger gene segments, PB1, PB2, and PA) was accom- the interaction of M1 and RNP remains unclear. To study plished by PCR using the same primer for RT and the universal primer the role by which M1 protein contributes to high-growth complementary to the conserved 5Ј end of genomic RNA. Partial properties, we began by comparing viral growth proper- dsDNA of PB1, PB2, or PA was amplified by using the same 3Ј end Ј ties of A/PR8/34 (laboratory-adapted high-growth strain), primer for RT and the 5 end primer corresponding to position 1238– 1259 of PB1, 2243–2267 of PB2, or 2104–2122 of PA. The genotypes of A/Nanchang/933/95 (a recent wild-type virus at low egg Resvir-9, A/PR8/34, and A/Nanchang/933/95 viruses were identified by passage), and Resvir-9 (a reassortant between A/PR8/34 analyzing the electrophoresis patterns of eight DNA fragments after and A/Nanchang/933/95). Table 1 shows the compara- digestion with unique restriction enzymes to each PCR product. BINDING OF M1 PROTEIN TO RNP CONTROLS VIRAL GROWTH 91

FIG. 1. Visualization of filamentous particle formation in MDCK cells infected with A/PR8/34, A/Nanchang/933/95, and Resvir-9 viruses. MDCK cells were infected with A/PR8/34, A/Nanchang/933/95, and Resvir-9 viruses at an m.o.i. of 3 plaque-forming units per cell. Immunofluorescence staining was performed at 4°C using anti-HA antibodies to A/PR8/34 and to A/Nanchang/933/95 and the cells were fixed in 3% paraformaldehyde. Viral filaments are visualized in (C), the cells infected with A/Nanchang/933/95, but are absent in cells infected with A/PR8/34 (A) or with Resvir-9 (B). Magnification, ϫ472. filamentous influenza virus particles at the infected cell by the velocity sucrose gradient centrifugation. After frac- surface can be visualized by using immunofluorescence tionation of the sucrose gradients, the proteins of the microscopy (Roberts and Compans, 1998; Roberts et al., viruses in the fractions were analyzed by SDS–PAGE. As 1998). To study the morphology of A/PR8/34, A/Nan- shown in Fig. 3, the viral proteins from A/PR8/34 (Fig. 3A) chang/933/5, and Resvir-9 viruses, the assembly of fila- were located mainly in fractions 3 to 5, with the peak in mentous or spherical particles at the surface of MDCK fraction number 4, indicating a slower migrating species cells infected by A/PR8/34, A/Nanchang/933/5, and compared to A/Nanchang/933/95 (Fig. 3C). In contrast, Resvir-9 were characterized by surface immunofluores- viral proteins from A/Nanchan/933/95 were confined to cence using antibodies to hemagglutinin. Cells infected fractions 5 and 6, indicating that they were faster migrat- with either A/PR8/34 virus (Fig. 1A) or with Resvir-9 virus ing. The distribution of viral proteins from Resvir-9 re- (Fig. 1B) showed the presence of viral antigens distributed sembled that of the particles of A/PR8/34 virus. These over the cell surface with intense fluorescence consis- results are consistent with the immunofluorescence tent with a spherical morphological phenotype. In con- data, which indicate that Resvir-9 obtained spherical trast, long filamentous particles were seen by immuno- phenotype by inheriting PB2, PB1, PA, M, and NS genes fluorescence of cells infected with A/Nanchang/933/95 from A/PR8/34. virus (Fig. 1C). The morphology of A/PR8/34, A/Nan- chang/933/5, and Resvir-9 viruses was also confirmed by A/PR8/34, A/Nanchang/933/95, and Resvir-9 viruses electron microscopy and shown in Fig. 2, indicating that differ in RNP-binding activity of M1 A/PR8/34 and Resvir-9 had predominantly spherical morphological phenotype (Figs. 2A and 2B) and that A/Nan- To relate growth properties of the viruses to the RNP- chang/933/95 virus had a filamentous phenotype (Fig. binding activity of the M1 proteins, we isolated M1/RNP 2C). These results suggest that HA, NA, and NP genes complexes from viral particles and performed a dissoci- from filamentous-parent A/Nanchan/933/95 are not a de- ation assay by treating M1/RNP complexes with varying terminant of filamentous morphology characteristics, salt concentration or with varying pH to release M1 since they were present in spherical Resviru-9 virus. protein from the complexes. Complexes containing RNP with un-disrupted M1 protein were separated from disrupted M1 by centrifugation through 20% glycerol. The A/PR8/34, A/Nanchang/933/95, and Resvir-9 viruses protein complexes were analyzed by SDS–PAGE and differ in migration in sucrose gradient shown in Fig. 4. Figure 4 shows a typical SDS–PAGE Filamentous particles are faster migrating species pattern of salt-disrupted M1/RNP complexes with NP than the spherical particles by velocity centrifugation in a migrating at approximately 56 kDa and M1 migrating at continuous 20–60% sucrose gradient (Roberts et al., approximate 27 kDa. With increasing salt concentration 1998). To confirm that the structures seen by light mi- (0.15 to 0.6 M), the amount of M1 retained in the M1/RNP croscopy corresponded to filamentous or spherical par- complexes decreased. Table 3 summarized the ratio and ticles, A/PR8/34, A/Nanchan/933/95, and Resvir-9 virus percentage of M1 to NP from calculating the density of particles propagated in eggs were purified and analyzed M1 and NP bands in Fig. 4. At 0.6 M salt concentration, 92 LIU, MULLER, AND YE

pH treatment. At pH 5, the ratio of M/NP from A/PR8/34 M1/RNP complexes was 0.20, and the ratio of M/NP from Resvir-9 was 0.21. However, the M/NP ratio of A/Nan- chang/933/95 M1/RNP complexes was 0.01. These results indicate that the M1 of high-growth A/PR8/34 has higher binding activity for RNP than the M1 of low-growth A/Nanchang/933/95. These results suggest that M1 is the control element in the M1/RNP complexes, because the reassortant, with M1 from A/PR8/34 and NP from A/Nanchang/933/95, exhibited dissociation properties similar to A/PR8/34.

DISCUSSION It has been reported that M1 and M2 proteins are involved not only in viral replication but also in viral morphogenesis and it has been suggested that the morphology of viral particle, such as filamentous vs spherical, may be related to the ratio of M1 and NP proteins (Roberts et al., 1998). To explore the role of M1 protein in

FIG. 2. Electron micrographs of negatively stained viral particles purified by sucrose gradient centrifugation. (A) A/PR8/34 virus; (B) Resvir-9 virus; (C) A/Nanchang/933/95. Samples were adsorbed onto Formvar-coated copper grids, negatively stained with 2% methylamine tungstate (Bio-Rad Microscience Division, Cambridge, MA), and examined in a LEO EM912 Omega transmission electron microscope. The bar represents 0.5 ␮m.

36% of M1 from A/PR8/34 remained associated with RNP (M1:NP ratio ϭ 0.40), compared with 3% of M1 from A/Nanchang/933/95 (M1:NP ratio ϭ 0.05), and 19% of M1 from Resvir-9 (M1:NP ratio ϭ 0.22). RNP-binding activity of M1 from high-growth and low-growth viruses was also compared under reduced pH conditions. Figure 5 shows a typical gel pattern of low-pH-disrupted M1/RNP complexes. With acidification (changing pH from 7 to 5), the amount of M1 proteins in M1/RNP complexes decreased. Table 3 summarizes the results of the ratio and percentage of M1 to NP from Fig. 5. At pH 5, 17% M1 from FIG. 3. Sedimentation profiles of A/PR8/34, Resvir-9, and A/Nan- A/PR8/34 remained associated with RNP, and 18% M1 chang/933/95 viruses. A/PR8/34 (A), Resvir-9 (B), and A/Nanchang/ 933/95 (C) viruses were analyzed by velocity gradient centrifugation in from Resvir-9 remained association with RNP, whereas 20–60% sucrose gradients. Gradients were fractioned and the proteins only 1% M1 from A/Nanchang/933/95 remained associ- of the viruses were analyzed by 12% SDS–PAGE and stained by 0.1% ated with RNP. The ratio of M/NP was also altered by low Coomassie brilliant blue. BINDING OF M1 PROTEIN TO RNP CONTROLS VIRAL GROWTH 93

FIG. 4. Effect of salt on M1/RNP association. Dissociation of M1 form RNP was carried out by exposing to NaCl at 0.15 M (0.15), 0.3 M (0.3), or 0.6 M (0.6). The protein components of RNPs (RNP) and virions (V) were analyzed on 12.5% SDS–PAGE and stained by 0.1% Coomassie brilliant blue. the relationship between replication efficiency and mor- 34 Ͼ Resvir-9 Ͼ A/Nanchang/933/95, and the growth phology, we have analyzed M1–RNP-binding activities of potential of these three viruses are also A/PR8/34 Ͼ viruses with a range of replication efficiency, including a Resvir-9 Ͼ A/Nanchang/933/95, we postulate that the reassortant originated between a high-growth and a low- RNP-binding activity of the M1 protein may be related to growth viruses. Our results suggest that the RNP-binding the growth properties of the viruses. activity of the M1 protein affects the replication efficiency Although, in these studies, we do not have direct of the viruses, and that the M1 that binds more strongly evidence to show that high-growth characteristics of to RNP is found in viruses that replicate more efficiently. Resvir-9, a reassortant of A/PR8/34 and A/Nanchang/ Resvir-9, which inherited P, M, and NS genes from 933/95 is due to the five “internal” genes (PB2, PB1, PA, A/PR8/34, but the rest of the genes from A/Nanchang/ M, and NS) of A/PR8/34, M gene has been believed to 933/95, shows intermediate growth and M1–RNP-bind- have a major effect on the morphology of the viral parti- ing properties. Previous studies suggest that association cles (Smirnov et al., 1991; Roberts et al., 1998). A reas- of M1 with RNP is through two separate mechanisms: sortant between A/duck/Hoshimin/014/78, which is com- M1–RNA interaction and M1–NP interaction (Wakefield posed of filamentous particles, and A/WSN/33, which is and Brownlee, 1989; Ye et al., 1999). Therefore, increased composed of spherical particles, lost its filamentous par- RNP binding of Resvir-9 may be due to the higher affinity ticle characteristics by inheriting only M gene from of inherited A/PR8/34-M1 to RNA and/or to NP of RNP A/WSN/33 (Smirnov et al., 1991). The higher growth char- complexes. Although RNP complex of Resvir-9 contains acteristics of Resvir-9 may result from the combination of P proteins from A/PR8/34 and NP protein from A/Nan- all five internal A/PR8/34 genes. The different growth chang/933/95, the RNP complex composes only one characteristics between A/PR8/34 and Resvir-9 may be copy of each of these A/PR8/34-P proteins but multi- due to HA, NA, and NP gene from A/Nanchang/933/95 copies of Nanchang-NP proteins, the effect of the P strain. The interaction between HA and M1 has been proteins from A/PR8/34 on association of M1 to RNP may suggested by studying the coexpressed HA and M1 be minimum. Because the RNP-binding activities of M1 proteins in cells (Enami and Enami, 1996). Recent studies proteins of M1/RNP complexes are in the order A/PR8/ of reassortment between avian influenza viruses and

TABLE 3 Effect of Salt and Low pH on Dissociation of M1 from RNP

PR8 Resriv-9 Nanchang

NaCl (M) V 0.15 0.3 0.6 V 0.15 0.3 0.6 V 0.15 0.3 0.6 M1/NP 1.10 0.90 0.86 0.40 1.18 0.92 0.76 0.22 1.90 1.10 0.42 0.05 (% of M1) 100 82 78 36 100 78 64 19 100 58 22 3 pHV 765V765V765 M1/NP 1.15 0.90 0.42 0.20 1.20 0.92 0.50 0.21 2.10 1.00 0.24 0.01 (% of M1) 100 78 37 17 100 77 42 18 100 48 11 Ͻ1

Note. Effect of salt and low pH on dissociation of M1 from RNP. The RNP-associated M1 and the ratio of M1 to NP were quantified by densitometry. The percentage of the binding activity of M1s to RNP was calculated by comparison of M1 protein to NP in virions and M1/RNP. The data were means of at least three independent experiments, and the standard deviation of each experimental point was below 10%. V, Virion. 94 LIU, MULLER, AND YE

FIG. 5. Effect of low pH on M1/RNP association. Dissociation of M1 from RNP was carried out in buffer containing 0.05 M NaCl at pH 7.0, 6.0, and 5.0. The protein components of RNPs (RNP) and virions (V) were analyzed on 12.5% SDS–PAGE and stained by 0.1% Coomassie brilliant blue. human influenza viruses suggest different capabilities association of M1 with RNP, resulting in efficient trans- between the HA of avian viruses and the M gene prod- location of RNP to cytoplasm, and may facilitate final ucts of human influenza A viruses (Scholtissek et al., particle assembly. 2002). Therefore, the different growth potentials between A/PR8/34 and Resvir-9 might be resulted from different MATERIALS AND METHODS capabilities between HA and M1. Taken together, although M gene alone has a major impact on viral mor- Virus and cells phology, growth characteristics may be resulted from the Influenza viruses A/PR8/34, A/Nanchang/933/95, and combination of other genes. Resvir-9 were propagated in the allantoic cavities of Previous observation (Robert et al., 1998) indicates 9-day-old embryonated eggs at 34°C for 2 days. Madin– that the percentage of NP is reduced in filamentous Darby canine kidney cells (MDCK) were grown and main- particles. Our comparative studies of the ratios of M1 tained in MEM supplemented with 5% fetal bovine se- and NP proteins in M1/RNP of A/PR8/34, Resvir-9, and rum. Infectivity titers of the viruses were determined by A/Nanchang/933/95 viruses (Fig. 3 and Table 3) also plaque assay in MDCK cells using an agar overlay sys- showed that A/PR8/34 and Resvir-9 had an approxi- tem containing 2 ␮g/ml trypsin. mately one-to-one ratio of M1 to NP, but A/Nanchang/ 933/95 had almost a two-to-one ratio of M1 in the M1/ Generation of reassortant influenza viruses RNP complex. Thus the ratio of M1/NP is relatively Resvir-9, reassortant between A/PR8/34 and A/Nan- higher in filamentous particles than that in spherical chang/933/95, was prepared as described previously particles. Since the incorporation of RNP complexes is (Offringa et al., 2000). Briefly, 9-day-old embryonated not reduced in filamentous formation (Robert et al., 1998; eggs were coinfected with A/PR8/34 and A/Nanchang/ Smirnov et al., 1991), “extra” M1 proteins might be incor- 933/95 at a 10Ϫ3 dilution. Allantoic fluids were collected, porated into filamentous particles to compensate the low mixed with ferret anti-A/PR8/34 antiserum, and subse- RNP-binding activity of M1. It has been also observed quently passed in eggs. Resvir-9 was isolated by end- that the RNP complex structure of filamentous particle is point dilution in eggs. The genotype of Resvir-9 was less dense than that of spherical particle (Ruigrok et al., determined by reverse transcription-polymerase chain 1985). Therefore, although there are more M1 proteins in reaction (RT-PCR) and restriction fragment length poly- the filamentous particle relative to RNP, M1 may be either morphism (RFLP) (Offringa et al., 2000). loosely associated with RNP or loosely binds to M1 itself. M1 protein plays an important role in particle assem- Immunofluorescence staining bly and viral replication. Both the dissociation of M1 from RNP in the early phase of the infection and the associ- Cultures of MDCK cells grown on glass coverslips ation of M1 and RNP in the late phase of the infection are were infected with influenza virus at an m.o.i. of 3 PFU required for sufficient viral replication. Our recent studies per cell and subsequently incubated in MEM containing demonstrate that vRNA and M1 together promote the 2% fetal bovine serum (FBS) at 33°C for 9 h. Infected self-assembly of influenza virus NP into the quaternary MDCK cells were fixed with freshly prepared 4% formal- helical structure of typical viral RNP. It is believed that the dehyde in phosphate-buffered saline (PBS) for 20 min at association of M1with RNP leads to translocation of RNP room temperature. Background staining was blocked from nucleus to cytoplasm. The contribution of higher with 3% powdered skim milk in PBS for 1 h. The cells RNP binding of M1 to higher growth characteristics of were then incubated at room temperature for 40 min A/PR8/34 and Resvir-9 may be partially due to a strong either with sheep anti-A/PR8/34 HA or with sheep anti- BINDING OF M1 PROTEIN TO RNP CONTROLS VIRAL GROWTH 95

A/Nanchang/933/95 HA. The cells were incubated with ACKNOWLEDGMENTS donkey anti-sheep IgG conjugated with fluorescence We thank Dr. Lewis Markoff and Dr. C. D. Atreya for critically reading and incubated for 40 min at room temperature. Washed the manuscript. We are especially grateful to Dr. Roland Levandowski coverslips were mounted in 90% glycerol and 10% PBS in for valuable discussions as well as for reading and improving the 3,4,5-trihydroxybenzoic acid N-propylester to prevent manuscript. photobleaching. Cellular distribution of immunofluores- REFERENCES cence was visualized by epifluorescent UV microscope. Allen, H., McCauley, J., Waterfield, M., and Gething, M. J. (1980). Influ- enza virus RNA segment 7 has the coding capacity for two polypep- Dissociation of M1 protein from M1/RNP complexes tides. Virology 107, 548–551. Baudin, F., Petit, I., Weissenhorn, W., and Ruigrok, R. W. (2001). In vitro Purified M1/RNP complexes of influenza A virus were dissection of the membrane and RNP binding activities of influenza obtained by disrupting influenza A viruses (0.5 mg/ml) in virus M1 protein. Virology 281, 102–108. disruption buffer containing 10 mM Tris–HCl (pH 7.0), 1% Bucher, D. J., Kharitonenkov, I. G., Zakomirdin, J. A., Grigoriev, V. B., Klimenko, S. M., and Davis, J. F. (1980). Incorporation of influenza Nonidet P-40, 0.05 M NaCl, and 1.25 mM dithiothreitol. virus M-protein into liposomes. J. Virol. 36, 586–590. After incubation in disruption buffer for 40 min at room Bui, M., Whittaker, G., and Helenius, A. (1996). Effect of M1 protein and temperature, M1/RNP complexes released from virions low pH on nuclear transport of influenza virus ribonucleoproteins. in the reaction mixture were pelleted by centrifugation J. Virol. 70, 8391–8401. Burnet, F. M., and Lind, P. E. (1957). Studies on filamentary forms of through 25% glycerol with a cushion of 50% glycerol in an influenza virus with special reference to the use of dark-ground- SW 55Ti rotor at 120,000 g for 60 min. The supernatant microscopy. Arch. Gesamte Virusforsch. 7, 413–422. fluid containing lipids and membrane proteins was dis- Compans, R. W., and Klenk, H. D. (1979). Viral membranes. In “Com- carded and the pellet containing the M1/RNP complexes prehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, Eds.), Vol. 13, pp. 293–407. Plenum Publishing Corp., New York. was resuspended in 10 mM Tris–HCl (pH 7.4). The anal- Enami, M., and Enami, K. (1996). Influenza virus hemagglutinin and ysis of dissociation of M1 from RNP was examined as neuraminidase glycoproteins stimulate the membrane association of follows: the suspension of M1/RNP complexes was in- the matrix protein. J. Virol. 70, 6653–6657. cubated in buffer containing 0.05 M NaCl and 0.05 M Gregoriades, A. (1980). Interaction of influenza M protein with viral lipid Na HPO adjusted to specific pH by addition of 0.1 M and phosphatidylcholine vesicles. J. Virol. 36, 470–479. 2 4 Heggeness, M. H., Smith, P. R., Ulmanen, I., Krug, R. M., and Choppin, citric acid. To study the effect of ionic strength on M1/ P. W. (1982). Studies on the helical nucleocapsid of influenza virus. RNP complex association, the salt concentration was Virology 118, 466–470. varied by adding NaCl to the reaction buffer. A 20-␮l Helenius, A. (1992). Unpacking the incoming influenza virus. Cell 69, aliquot of the RNP-M1-detergent suspension was centri- 577–578. ␮ Huang, X., Liu, T., Muller, J., Levandowski, R. A., and Ye, Z. (2001). Effect fuged through a 150- l cushion of 20% sucrose at 10,000 of influenza virus matrix protein and viral RNA on ribonucleoprotein rpm for 15 min in a 0.5-ml Eppendorf tube, and the formation and nuclear export. Virology 287, 405–416. resulting pellet was collected. The protein composition Kilbourne, E. D., and Murphy, J. S. (1960). Genetic studies of influenza of the RNPs was analyzed by electrophoresis in 12.5% virus. 1. Viral morphology and growth capacity as exchangeable genetic traits. Rapid in ovo adaptation of early passage Asian strain polyacrylamide–SDS gels at non-reducing condition and isolated by combination with PR8. J. Exp. Med. 111, 387–406. stained with Coomassie blue. The amount of M1 and NP Lamb, R. A., and Choppin, P. W. (1983). The gene structure and repli- proteins was determined by densitometry of proteins. cation of influenza virus. Ann. Rev. Biochem. 52, 467–506. Lamb, R. A., Holsinger, L. J., and Pinto, L. H. (1994). The influenza A virus M2 ion channel protein and its role in the influenza virus life cycle. In Sucrose gradient analysis of virus particles “Receptor-Mediated Virus Entry into Cells” (E. Wimmer, Ed.), pp. 303–321. Cold Spring Harbor Press, Cold Spring Harbor, NY. Sucrose gradient analysis of viral particles was Lamb, R. A., Zebedee, S. L., and Richarson, C. D., (1985). Influenza virus adapted from Roberts et al. (1998), with minor modifica- M2 protein is an integral membrane protein expressed on the in- tion. Briefly, influenza viruses, A/PR8/34, A/Nanchan/933/ fected-cell surface. Cell 40, 627–633. 95, and Resvir-9, were propagated in embryonated eggs, Martin, K., and Helenius, A. (1991). Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export concentrated, and purified by banding in 15 to 60% su- and inhibits import. Cell 67, 117–130. crose gradients at 100,000 g for 90 min at 4°C. The viral Murti, K. G., Webster, R. G., and Jones, I. M. (1988). Localization of RNP bands were collected and pelleted through 25% sucrose polymerases on influenza viral ribonucleoprotein by immunogold cushions at 100,000 g for 90 min at 4°C. The pelleted labeling. Virology 164, 562–566. Offringa, D. P., Tyson-Medlock, V., Ye, Z., and Levandowski, R. A. (2000). viruses were resuspended slowly overnight at 4°C in the A comprehensive systematic approach to identification of influenza A buffer containing 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, virus genotype suing RT-PCR and RFLP. J. Virol. Methods 88, 15–24. and 1 mM EDTA. Viruses were then fractionated by Patterson, S., Gross, J., and Oxford, J. S. (1988). The intercellular distri- velocity centrifugation in a continuous 20–60% sucrose bution of influenza virus matrix protein and nucleoprotein in infected gradient at 100,000 g for2hat4°C. Virus fractions cells and their relationship to hemagglutinin in the plasma membrane. J. Gen. Virol. 69, 1859–1872. collected from the top to the bottom were used to analyze Rees, P. J., and Dimmock, N. J. (1982). Kinetics of synthesis of influenza viral proteins in12% SDS–PAGE. virus ribonucleoprotein structures. J. Gen. Virol. 59, 403–408. 96 LIU, MULLER, AND YE

Roberts, P. C., and Compans, R. W. (1998). Host cell dependence of viral Schulze, I. T. (1972). The structure of influenza virus. II. A model based morphology. Proc. Natl. Acad. Sci. USA 95, 5746–5751. on the morphology and composition of subviral particles. Virology 47, Roberts, P. C., Lamb, R. A., and Compans, R. W. (1998). The M1 and M2 181–196. proteins of influenza A virus are important determinants in filamen- Smirnov, Y. A., Kuznetsova, M. A., and Kaverin, N. V. (1991). The genetic tous particle formation. Virology 240, 127–137. aspects of the influenza virus filamentous particle formation. Arch. Robertson, B. H., Bennett, C. J., and Compans, R. W. (1982). Selective dan- Virol. 118, 279–284. sylation of M protein within intact influenza virus. J. Virol. 44, 871–876. Sugrue, R. J., and Hay, A. J. (1991). Structural characteristics of the M2 Ruigrok, R. W. H., and Baudin, F. (1995). Structure of influenza virus ribo- protein of the influenza A viruses: Evidence that it forms a tetrameric nucleoprotein particles. II. Purified RNA-free influenza virus ribonucleo- channel. Virology 180, 617–624. protein form structures that are indistinguishable from the intact influ- Wakefield, L., and Brownlee, G. G. (1989). RNA-binding properties of enza virus ribonucleoprotein particles. J. Gen. Virol. 76, 1009–1014. influenza A virus matrix protein M1. Nucleic Acids Res. 17, 8569– Ruigrok, R. W. H., Krijgsman, P. C. J., de Roude-Verloop, F. M., and de 8680. Jong, J. C. (1985). Natural heterogenicity of shape, infectivity and Ye, Z., Pal, R., Fox, J. W., and Wagner, R. R. (1987). Functional and protein composition in an influenza A (H3N2) virus preparation. Virus antigenic domains of the matrix (M1) protein of influenza virus. Res. 3, 69–76. J. Virol. 61, 239–246. Scholtissek, C., Stech, J., Krauss, S., and Webster, R. G. (2002). Coop- Ye Z, Liu, T., Offringa, D. P., McInnis, J., and Levandowski, R. A. (1999). eration between the hemagglutinin of avian viruses and the matrix Association of influenza virus matrix protein with ribonucleoproteins. protein of human influenza A viruses. J. Virol. 76, 1781–1786. J. Virol. 73, 7467–7473.