Journal of General Virology (2009), 90, 2751–2758
DOI 10.1099/vir.0.014316-0
Influenza A virus M1 blocks the classical complement pathway through interacting with C1qA Junjie Zhang,1,2 Gang Li,1,2 Xiaoling Liu,2,3 Zengfu Wang,2,3 Wenjun Liu3 and Xin Ye1 1
Correspondence
Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
Wenjun Liu
[email protected] Xin Ye
2
Graduate University of Chinese Academy of Sciences, Beijing 100101, PR China
[email protected]
3
Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
Received 11 June 2009 Accepted 30 July 2009
The matrix (M1) protein of influenza A virus is a conserved multifunctional protein that plays essential roles in regulating the viral life cycle. This study demonstrated that M1 is able to interact with complement C1qA and plays an important inhibitory function in the classical complement pathway. The N-terminal domain of M1 protein was required for its binding to the globular region of C1qA. As a consequence, M1 blocked the interaction between C1qA and heat-aggregated IgG in vitro and inhibited haemolysis. It was shown that M1 protein prevented the complementmediated neutralization of influenza virus in vitro. In addition, studies on mice indicated that the administration of M1 could promote a higher virus propagation rate in lung and shortened survival of mice infected with the virus. Taken together, these results suggest strongly that the M1 protein plays a critical role in protecting influenza virus from the host innate immune system.
INTRODUCTION The genome of influenza A virus is composed of eight segments of single-stranded RNA, which exist as a viral ribonucleoprotein (vRNP) complex with nucleocapsid protein (NP) and RNA-dependent RNA polymerase (Neumann et al., 2004). vRNP is surrounded by matrix protein 1 (M1), which is the most abundant protein in influenza virus particles (Nayak et al., 2004). Influenza A virus M1 protein is relatively conserved and consists of two globular regions (aa 1–164 and 165–252). The structure of the N-terminal fragment of the M1 protein determined by X-ray crystallography analysis shows the existence of nine helices and eight loops (Arzt et al., 2001; Harris et al., 2001; Sha & Luo, 1997), whilst the structure of the C-terminal domain of M1 is not yet available. It is widely accepted that M1 plays a significant role in many aspects of the virus life cycle. M1 interacts with RNA and RNPs (Baudin et al., 2001; Bui et al., 1996; Elster et al., 1997; Huang et al., 2001; Wakefield & Brownlee, 1989; Watanabe et al., 1996; Ye et al., 1999) and viral envelope protein (Ali et al., 2000; Barman et al., 2001), and is involved in transcription inhibition (Baudin et al., 2001; Elster et al., 1997; Watanabe et al., 1996), RNP nuclear import/export and the budding A supplementary figure showing anti-M1 antibody detection in human and mouse sera is available with the online version of this paper.
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process (Akarsu et al., 2003; Bourmakina & Garcı´a-Sastre, 2005; Nayak et al., 2004). M1 expressed on the infected cell surface is responsible for cross-reactive recognition by cytotoxic T lymphocytes (Braciale, 1977; Reiss & Schulman, 1980). Several other studies have also suggested that some M1 mutations affect virus particle morphology (Burleigh et al., 2005) and are involved in virus–host protein interactions (Nagata et al., 2008). Several host proteins have been found to associate with M1, including the globular domain of the histone octamer (Garcia-Robles et al., 2005), heat-shock protein 70 (Watanabe et al., 2006) cytoskeletal elements (Avalos et al., 1997), the cellular receptor of activated C kinase 1 (Reinhardt & Wolff, 2000) and caspase 8 (Zhirnov et al., 2002). These interactions imply a broad range of roles of biological significance, such as vRNP export and viral morphogenesis. Extracellular signal-regulated kinase (ERK), downstream of the Ras-activated factor Raf/MEK/ ERK pathway, phosphorylates M1. When cells infected with influenza virus are treated with a MEK-specific inhibitor, NP and vRNP complexes accumulate in the nucleus (Pleschka et al., 2001). A recent report indicated that cyclophilin A interacts with M1 and impairs early virus replication (Liu et al., 2009). The interactions between M1 and host proteins are critical for viral propagation, but it is 2751
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unknown whether M1 protein has any effects on host immune responses. A number of studies have focused on the mechanism of influenza virus neutralization by serum. It had been found that human serum can neutralize strain A/WSN/33 virus efficiently through specific antibodies and the C1q–C4mediated complement pathway (Beebe et al., 1983). In a study carried out with mouse serum, researchers found that C1q but not C3 was needed for virus neutralization and that a heat-resistant factor also contributed to virus neutralization (Mozdzanowska et al., 2006). A recent study indicated that natural IgM could restore virus neutralization activity to antibody-deficient serum and that the mechanism of virus neutralization by natural IgM was associated with virion aggregation, which was dependent on C1q–C4 of the complement pathway, but not on C5mediated viral lysis (Jayasekera et al., 2007). Taken together, these data indicate the involvement of complement, or at least C1q, in influenza virus neutralization by serum. To understand the function of M1 in more detail, we took the approach of yeast two-hybrid screening to identify host proteins that interact with M1. In the present study, the complement component C1qA was identified as an M1binding protein. Further analysis showed that M1 interacted with the globular region of C1qA. The globular region of C1qA is the binding domain for the antigen– antibody complex. Our data suggest that the M1 protein can efficiently suppress the classical pathway and protect virus from being neutralized. We found that mice administrated with M1(1–170) had enhanced influenza virus propagation in lung and shortened survival rate when compared with mice treated with M1(171–252). Taken together, our results indicate that influenza virus M1 protein has an important role in protecting the virus from the host innate immune system via its interaction with complement C1qA.
METHODS Cell lines, viruses and antibodies. Madin–Darby canine kidney
(MDCK) cells and human embryo kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; PAA). Influenza virus strain A/WSN/33 (H1N1) was generated by reverse genetics (Neumann et al., 1999) and propagated in MDCK cells. Mouse anti-M1 monoclonal antibody was prepared as described previously (Koestler et al., 1984). Rabbit anti-human C1qA and rabbit anti-NP polyclonal antibodies were generated by immunizing rabbits with glutathione S-transferase (GST)–C1qA(24–70) fusion and Histagged NP protein, respectively. The antibodies were affinity purified with antigen-conjugated agarose. Monoclonal anti-Myc (9E10) and anti-His antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-FLAG (M2) antibody was purchased from Sigma. Construction of plasmids. Full-length and truncated forms of M1
were subcloned into pET30a (Novagen). Full-length C1qA and truncated forms were subcloned into pGEX-6P-1(Pharmacia). Fulllength M1, C1qA and truncated C1qA were subcloned into pENTR 2752
vector (Invitrogen) and transferred into pDEST-Myc or pDESTFLAG expression vector (Invitrogen) using an LR Clonase enzyme mix kit (Invitrogen) following the manufacturer’s instructions. Yeast two-hybrid system. A Matchmaker Two-hybrid System 3
(Clontech) was used to screen host proteins that interacted with M1. In brief, the bait construct pGBKT7-M1 and a human kidney cDNA library in pACT2 (prey) were co-transformed into yeast strain AH109. Transformants were selected for growth on medium lacking His, Leu and Trp (His–/Leu–/Trp–). The colonies were then transferred to Ade–/His–/Leu–/Trp– plates containing X-Gal. Blue colonies were selected and cultured in Ade–/His–/Leu–/Trp– broth and lysed for plasmid extraction. The plasmids were amplified and the target insertions were verified by sequencing. To confirm interaction between M1 and host proteins, the two yeast expression plasmids were co-transformed into yeast strain SFY-526 and b-galactosidase assays were performed according to manufacture’s instruction. Protein purification, and GST and His pull-down assays. GST
fusion and His-tagged proteins were purified with Sepharose 4B– glutathione (Pharmacia) and Ni-NTA (Qiagen), respectively. For the GST pull-down assay, 5 mg GST or GST–C1q fusion protein bound to Sepharose 4B–glutathione was incubated with purified recombinant M1 or FLAG–M1 293T cell lysate at 4 uC for 4 h. The beads were washed five times with PBS with 0.1 % Triton X-100, and bound protein was eluted by boiling in SDS loading buffer and subjected to Western blot analysis. For the His pull-down assay, 5 mg Ni-NTAbound His-tagged full-length and truncated M1 proteins were incubated with 1 mg recombinant GST–C1qA in His pull-down buffer (200 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 1 mM MgCl2, 0.1 % Triton X-100, 20 mM immidazole, 1 mg BSA ml21) at 4 uC for 2 h. After incubation, the beads were centrifuged and washed with pull-down buffer. Bound proteins were eluted with 50 ml pull-down buffer supplemented with 200 mM imidazole and analysed by Western blot analysis with rabbit polyclonal anti-C1qA antibody. Co-immunoprecipitation. 293T cells were transfected with FLAGtagged and Myc-tagged plasmid. At 36 h post-transfection, cells were washed with cold PBS and lysed in lysis buffer [1 % Triton X-100, 150 mM NaCl, 20 mM HEPES (pH 7.5), 10 % glycerol, 1 mM EDTA] with protease inhibitor cocktail (Roche). The lysates were immunoprecipitated with anti-FLAG beads at 4 uC for 4 h. The beads were washed five times with lysis buffer. Bound proteins were eluted by boiling with SDS loading buffer for 10 min and subjected to Western blot analysis with anti-Myc monoclonal antibody. ELISA assays. ELISA was performed using Maxisorb plates (Nunc) coated with proteins diluted in coating buffer [100 mM Na2CO3/ NaHCO3 (pH 9.6)] at 4 uC overnight followed by blocking with 10 % FBS in PBS for 2 h at 37 uC. All subsequent steps were performed in PBS containing 0.05 % Tween 20 unless otherwise indicated, and each step was followed by three washes with PBS/0.05 % Tween 20. The enzymic activity of horseradish peroxidase (HRP) was measured by the addition of 2,29-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (Sigma) and H2O2. A405 was measured using a microplate absorbance reader (Tecan Sunrise).
To assess the effect of M1 on C1q and IgG binding, plates were coated with 5 mg purifed GST–C1qA(23–245) ml21. Heat-aggregated IgG (100 ml, 2 mg ml21; Zhongshan Goldenbridge Biotechnology) was added in the presence of M1 at various concentrations. IgG bound to C1qA was detected with HRP-conjugated goat anti-human IgG antibody. A405 (blank) was the absorbance obtained when no IgG was added, whilst A405 (100 %) was the absorbance obtained when IgG was added in the absence of M1. The percentage inhibition was calculated using the formula 12[A405 (measurement)2A405 (blank)]/ [(A405 (100 %)2A405 (blank)]. Journal of General Virology 90
M1 blocks the C1qA-mediated complement pathway To detect anti-M1 antibody in serum, plates were coated with 5 mg M1(1–170) ml21. The indicated amount of serum was added and bound antibody was detected with HRP-conjugated goat anti-mouse antibody. Virus neutralization assays. Human serum was obtained from a healthy volunteer and stored at 280 uC. Influenza virus A/WSN/33 (50 ml, 106 p.f.u. ml21) was mixed with 10 ml human serum or heatinactivated serum in the presence of 20 mM M1 or BSA at 37 uC for 30 min. The samples were analysed immediately by plaque assay. Plaque assay. MDCK cell monolayers in 35 mm dishes were washed
with PBS and serial dilutions of virus were adsorbed to the cells for 2 h. Unadsorbed virus was removed by washing with serum-free DMEM, and the cell monolayers were then overlaid with DMEM supplemented with 3 % low-melting-point agarose and 2 mg TPCKtreated trypsin (Sigma) ml21. After 3 days of incubation, visible plaques were counted and virus titres were calculated. All data were expressed as the mean of triplicate samples.
washed and resolved by SDS-PAGE. M1 proteins were detected in the GST–C1qA pull-down materials by Western blotting with anti-FLAG antibody, indicating that GST– C1q fusion protein could interact with FLAG–M1 from the cell lysate (Fig. 1a). In a similar experiment, GST–C1qA fusion protein was found to bind to purified recombinant His–M1 fusion protein but not to GST alone (Fig. 1b). To examine the interaction of M1 and C1qA in cells, FLAGtagged M1 and Myc-tagged C1qA were transiently expressed in 293T cells and cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Myc antibody. The result showed that FLAG–M1 indeed interacted with Myc–C1qA (Fig. 1c). Furthermore, FLAG–C1qA expressed in cells bound to M1 but not to NP, indicating that C1qA interacts specifically with M1 (Fig. 1d).
Haemolytic assay. To determine the haemolytic activity of the classical component activity, sheep red blood cells (SRBCs) were sensitized using rabbit anti-SRBC polyclonal antibody. Antibodysensitized SRBCs (10 ml) were then incubated with 10 ml human serum in the presence of the indicated concentration of M1(1–170) or BSA at a final volume of 1 ml PBS at 37 uC for 30 min and then centrifuged at 2000 g for 1 min. The A450 of the supernatant was measured against a reagent blank in which SRBCs were incubated with PBS. The percentage haemolysis was calculated by normalizing against the absorbance of a mixture of red blood cells and distilled water, which showed complete haemolysis. Mice and virus infection. Four-week-old specific-pathogen-free
BALB/c mice were used in these studies. Experiments were performed in accordance with institutional guidelines. M1 administration and mice infection were performed according to a modification of a standard procedure (Jayasekera et al., 2007). Briefly, mice were anaesthetized with ether and then administration with 200 ml M1(1– 170) or M1(171–252) (1 mg ml21) via the tail vein. The mice were then immediately infected intranasally with 25 ml influenza virus (26105 p.f.u. ml21). At 3 days post-infection, three mice from both groups were sacrificed and virus titres in the lungs were determined by plaque assay. Mice were examined daily for survival.
RESULTS Influenza A virus M1 protein interacts with C1qA To explore the function of the M1 protein, we attempted to search for cellular proteins that interacted with it. We screened a human kidney cDNA library by using a yeast two-hybrid system. The full-length M1 coding sequence was cloned into the pGBKT7 vector to generate pGBKT7M1 as bait. The bait construct was used to co-transfect yeast strain AH109 with the cDNA library in pACT2, followed by screening on Leu2 Trp2 His2 plates. This screening yielded a cDNA clone encoding the complement fragment C1qA, suggesting that C1qA peptide interacts with M1. To confirm the interaction between M1 and C1qA, GST pull-down assays were carried out. GST and GST–C1qA fusion proteins bound to Sepharose 4B– glutathione were incubated with cell lysates prepared from pDEST-FLAG-M1-transfected 293T cells. Complexes were http://vir.sgmjournals.org
Fig. 1. Influenza virus matrix protein M1 interacts with C1qA. (a, b) GST pull-down assay. Cell lysates from pDEST-FLAG-M1 transfected 293T cells or purified His–M1 proteins were incubated with GST and GST–C1qA, respectively. M1 protein associated with C1qA was analysed by Western blotting with anti-FLAG antibody (a) and anti-His antibody (b). (c, d) Co-immunoprecipitation analysis. (c) 293T cells were transfected with pDEST-MycC1qA and pDEST-FLAG-M1 or pDEST-FLAG-M1 alone. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Myc antibody. (d) 293T cells were transfected with pDEST-FLAG-C1qA or pcDNA3.1 and then infected with A/WSN/33 virus (m.o.i. of 1) for 12 h. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-M1 or anti-NP antibody. 2753
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To characterize further the interaction between M1 and C1qA, we attempted to map the region of M1 required for interaction with the complement fragment. A series of truncated mutants of M1 was constructed (Liu et al., 2009) (Fig. 2a) and applied to a His pull-down assay. As shown in Fig. 2(b), the N-terminal aa 1–170 of M1 were sufficient to bind C1qA. A GST pull-down assay also showed that GST– C1q bound to M1(1–170) but not M1(171–252) (Fig. 2c), indicating that M1(1–170) was required for its binding to C1qA. A previous study showed that the C1qA globular head region plays an important role in the classical complement pathway (Kaul & Loos, 1995). In the present study, we demonstrated that M1(1–170) interacted with the C1qA globular head region (aa 110–245) but not with C1qA(1–109) by a GST pull-down assay (Fig. 3a). Coimmunoprecipitation analysis also indicated that fulllength M1 interacted with the C1qA globular region (Fig. 3b). As the C1qA globular region plays an important role in the classical complement pathway, our results implied that M1 may subvert the host classical complement system during virus infection. M1 blocks C1qA-mediated classical complement activity C1qA is the first subcomponent of the C1 complex in the classical complement pathway and plays a key role in recognizing immunoglobulin through its globular head region (Kishore & Reid, 2000). Therefore, we hypothesized that the binding of M1 to the globular head region of C1qA could impair complement activity. To assess the effect of
M1 on the interaction between C1qA and IgG, we first used an ELISA to examine the changes in binding of IgG to C1qA coated on plates with an increasing M1 protein concentration. As shown in Fig. 4, M1 was found to block the binding of IgG to C1qA in a dose-dependent manner. This suggested that M1 may be a potent inhibitor of the classical complement pathway. We then used haemolytic assays to measure the classical pathway complement activation and the effect of M1 protein on this. The data showed that M1 blocked the complement-dependent haemolysis of SRBCs in a dose-dependent manner, indicating that M1 could interfere efficiently with the classical complement pathway (Fig. 5). M1 counteracts neutralization of virus by human serum It has been shown that influenza virus can be neutralized in vitro by the classical complement pathway (Beebe et al., 1983; Jayasekera et al., 2007; Mozdzanowska et al., 2006). As our results showed that M1 interacted with C1qA and blocked classical pathway activation, we asked whether M1 was able to protect influenza virus from being neutralized by serum. To address this question, influenza virus A/ WSN/33 was incubated with normal or heat-inactivated human serum in the presence or absence of M1 protein for 30 min. After treatment, the virus was titrated by plaque assay. As shown in Fig. 6, when human serum was preincubated with M1 protein, virus neutralization was partly counteracted as the virus titre was fourfold higher than that in the control. However, there was no significant difference
Fig. 2. M1(1–170) binds C1qA. (a) Diagram of the series of deletion mutants of M1 and their interaction with C1qA, determined by a His pull-down assay and/or by immunoprecipitation (IP). ND, Not determined. (b) His pulldown assay. His-tagged recombinant M1 and its deletion mutants were incubated with GST–C1qA. Bound proteins were eluted and analysed by Western blotting with anti-C1qA antibody. CBB, Coomassie brilliant blue staining. (c) His–M1(1–170) or M1(171–252) were incubated with GST or GST–C1qA. Proteins associated with C1qA were analysed by Western blotting analysis and detected with anti-His or anti-M1 antibody. 2754
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Fig. 3. M1 protein binds to the C1qA globular region. (a) GST pull-down assay. His–M1(1– 170) were incubated with GST or GST– C1qA(1–109) or GST–C1qA(110–245) beads. After washing, the bound proteins were subjected to immunoblotting with anti-His antibody. (b) Co-immunoprecipitation. 293T cells were transfected with pDEST-MycC1qA(110–245) with pDEST-FLAG-M1 or pcDNA3.1 as control. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Myc antibody.
in the virus titre with or without M1 when the serum was heat-inactivated. Taken together, these results indicated that M1 can counteract the factors in serum that neutralize influenza virus and are sensitive to heat inactivation. It is likely that the complement components in the serum are the factors involved in neutralizing the virus and that this neutralization can be inhibited in the presence of M1 proteins. Mice become more susceptible to virus infection after administration with M1 Finally, we asked whether M1 administration facilitated influenza virus propagation in vivo. To address this
question, BALB/c mice were administrated with 200 mg M1(1–170) or M1(171–252) and immediately infected intranasally with influenza virus (56103 p.f.u.). Virus titres in the lungs of these mice were measured at 72 h postinfection. As seen in Fig. 7(a), the virus titre in M1(1–170)administrated mice was approximately threefold higher than that in M1(171–252)-treated mice. Consistent with these results, the survival period of mice administrated M1(1–170) was on average reduced by 1 day compared with mice administrated with M1(171–252) (Fig. 7b). As only M1(1–170), which binds to C1qA, is known to make mice more vulnerable to viral infection, this suggested that M1 may facilitate virus replication by counteracting virus neutralization by the C1qA-mediated complement pathway.
DISCUSSION
Fig. 4. M1 inhibits the binding of C1qA to IgG. Purified recombinant GST–C1qA(23–245) was coated on the plate, followed by a blocking step and addition of heat-aggregated human IgG and purified M1. IgG bound to C1qA was estimated using HRP-conjugated goat anti-human IgG antibody. The percentage inhibition was calculated using the formula described in Methods. http://vir.sgmjournals.org
Viruses have developed numerous strategies to counteract the classical complement system. Many virus proteins have been found to regulate complement activation and their functions to cover almost every step of the classical complement activation pathway (Favoreel et al., 2003). Herpes simplex virus expresses Fc receptors on the surface of infected cells, which inhibits efficient activation of antibody-dependent complementary components (Frank & Friedman, 1989). The VCP protein of vaccinia virus and SPICE protein of variola virus have been shown to inactivate C3b and C4b as co-factor I activity. Herpesvirus saimiri CCPH protein effectively inhibits C3 convertase activity and reduces cell-surface deposition of C3b. Cowpox virus IMP protein limits macrophage infiltration by downregulating complement proteins C3a, C4a and C5a (Howard et al., 1998; Kotwal et al., 1998). Glycoprotein C of herpes simplex virus mediates inhibition of C5 binding to C3b (Friedman et al., 1984; Hung et al., 1992, 1994) and the HVS-15 protein of herpesvirus saimiri inhibits complement activity after C3b deposition, indicating terminal complement inhibition (Rother et al., 1994). However, so far, little is known about whether influenza 2755
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Fig. 5. Haemolytic assay. SRBCs were sensitized using rabbit anti-SRBC antibodies. Fresh normal human serum was used as a complement source. Antibody-sensitized SRBCs were incubated with human serum in the presence of the indicated concentrations of M1 (h) or BSA (X) at 37 6C for 30 min. Haemolysis was assessed by measuring A450.
virus has developed a strategy to suppress the classical complement pathway. Previous studies have focused mainly on the mechanism of virus neutralization by complement. Human serum was found to neutralize influenza virus efficiently by an antibody-dependent mechanism, and complement components (C1, C3 and C4) were also indispensable for neutralization, although membrane attack pathways were not needed for the process (Beebe et al., 1983). A recent study demonstrated that, in mice, natural antibody rather than specific antibody mediated neutralization of influenza virus. It had been confirmed that C1, C3 and C4 are required in the process (Jayasekera et al., 2007). It should be noted that all of these previous studies are consistent
Fig. 7. M1(1–170) makes mice more susceptible to virus infection. Mice (nine per group) were administrated M1(1–170) or M1(171–252) and infected with virus intranasally. (a) Three mice from each group were sacrificed at 72 h post-infection. The lungs were collected to measure virus titre by plaque assay. (b) Infected mice were observed daily and the percentage survival of virus-infected mice was plotted against the number of days post-infection. X, M1(1–170); h, M1(170–252).
with C1 being indispensable in the neutralization of influenza virus by complement. The present study also confirmed that the virion could be neutralized by complement (Fig. 6). Taken together, these results indicated that the influenza virion can be neutralized efficiently by complement and that neutralization is dependent on complement C1. Influenza virus may develop some strategies to block this complement C1dependent neutralization. A recent study reported that human astrovirus coat protein inhibits complement activation via C1 (Bonaparte et al., 2008), which provides a good example of virus targeting complement by binding with C1.
Fig. 6. M1 inhibits complement-dependent virus neutralization. Fresh normal human serum was used as a complement source. Virus A/WSN/33 was incubated with normal human serum or heat-inactivated serum and M1(1–170) fragment (filled bars) or BSA (empty bars). Virus titres in the serum samples were estimated by plaque assay. 2756
The present study demonstrated that M1 may play a similar role in counteracting complement via C1. Firstly, M1 is highly abundant in influenza virus-infected cells and it is plausible that, at the late phase of virus infection, some host cells are so heavily destroyed that any matrix protein that is not assembled into virus particles is released and counteracts the complement system to protect new virus particles before they can attach to other cells. Secondly, Journal of General Virology 90
M1 blocks the C1qA-mediated complement pathway
anti-M1 antibodies were detected in human serum and the serum of virus-infected mice (see Supplementary Fig. S1, available in JGV Online), indicating that M1 was present in body fluids of the host following influenza virus infection, probably due to the non-specific release of M1 as a result of cell necrosis. These results indicate that M1 can bind to the C1qA globular region to efficiently reduce the interaction between C1qA and IgG and inhibit classical complement activity-dependent haemolysis. These results suggest that M1 is a potential complement pathway inhibitor by disrupting C1q-dependent classical complement activity. C1q is a key component of the classical complement pathway and acts as a recognition molecule that interacts with antibody–antigen complexes to activate the complement cascade. Some studies have shown that specifically designed peptides that bind C1q can inhibit the complement pathway (Roos et al., 2001). According to the present results, the M1 protein may act as a virusdesigned complement inhibitor that is abundantly expressed during virus infection and released during infected cell necrosis to protect newly assembled virus particles from being neutralized by complement. The present study indicates that influenza A virus M1 protein blocks the complement pathway through its interaction with C1qA. This finding may help to elucidate the connection between the innate immune system and influenza virus, and explain how influenza virus counteracts the innate immune system.
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ACKNOWLEDGEMENTS We thank Y. Zan (Institute of Microbiology, Chinese Academy of Sciences) for experimental help. We are grateful to W. Shou (Indiana University School of Medicine) for his critical reading of the manuscript. This work was supported by grants from the Ministry of Science and Technology of China (2007DFC30240, 2004BA519A64) and the Chinese Academy of Sciences Innovation projects (KSCX2-YW-N-054).
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