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• W2925325810 abstract "•Complement components C1 and C4 mediate potent neutralization of adenovirus•Deposition of C4b on the viral capsid inactivates capsid disassembly•C4 exerts direct antiviral functions independent from its role as a C3-convertase•C4 antiviral functions synergize with TRIM21-mediated virus neutralization The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r2C1s2) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy. The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r2C1s2) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy. The complement system encompasses >20 serum proteins that attack circulating pathogens, labeling them for destruction and promoting an inflammatory immune response. Complement can label pathogens in three ways, referred to as the alternative, mannose-binding lectin, and classical pathways. Of the three, the classical pathway is the most broadly effective, as it uses anti-pathogen antibodies and thus has the potential not only to recognize any target but also to direct complement to a specific pathogen during infection. There are two key properties that make the complement system so efficient at coating pathogens. First, it functions as an enzymatic cascade where preceding components catalyze the accumulation of subsequent proteins, thereby amplifying the response. Second, complement components couple themselves to a surface covalently, meaning that association with a pathogen is thermodynamically irreversible and long lived. At the core of the complement cascade are three paralogous proteins, C3, C4, and C5—the former two of which undergo covalent attachment. Under the classical pathway, pathogen-bound IgM or IgG first recruits the C1 complex (C1qC1r2C1s2), which cleaves C4 to expose its thioester and drive pathogen coupling. Cleaved C4b forms a complex with C2a called the “C3 convertase,” which proteolyzes C3, exposing its thioester and catalyzing surface deposition and resulting in C3b opsonization. Since C3b opsonization is vital for downstream processes such as membrane-attack-complex formation and phagocytosis, C3 is the most extensively studied complement component. In contrast, C4 has been studied almost exclusively in its context as a convertase for C3, and any distinct mechanisms that it may have remain largely unexplored. Partly, this is because as an upstream component, defects to C4 may have a knockon effect on C3. However, human genetics suggest that C4 has important immune roles unrelated to its C3 convertase function. Approximately 75% of patients with C4 deficiency have systemic lupus erythematosus (SLE), whereas <10% of those deficient in C3 have lupus-like symptoms (Carneiro-Sampaio et al., 2008Carneiro-Sampaio M. Liphaus B.L. Jesus A.A. Silva C.A. Oliveira J.B. Kiss M.H. Understanding systemic lupus erythematosus physiopathology in the light of primary immunodeficiencies.J. Clin. Immunol. 2008; 28: S34-S41Crossref PubMed Scopus (67) Google Scholar). The importance of C4 in immunity can also be inferred from viral mechanisms of antagonism. Vaccinia complement-control protein mimics C4-binding protein, thereby accelerating C4 removal from the viral surface (McKenzie et al., 1992McKenzie R. Kotwal G.J. Moss B. Hammer C.H. Frank M.M. Regulation of complement activity by vaccinia virus complement-control protein.J. Infect. Dis. 1992; 166: 1245-1250Crossref PubMed Scopus (137) Google Scholar), while flavivirus NS1 protein binds C4 and recruits C1s, causing C4 cleavage in solution and thus reducing surface deposition (Avirutnan et al., 2010Avirutnan P. Fuchs A. Hauhart R.E. Somnuke P. Youn S. Diamond M.S. Atkinson J.P. Antagonism of the complement component C4 by Flavivirus nonstructural protein NS1.J. Exp. Med. 2010; 207: 793-806Crossref PubMed Scopus (205) Google Scholar). Complement neutralization has so far only been demonstrated for enveloped viruses, with C4 exerting neutralization activity independently from C3 in the cases of equine arteritis virus (EAV) and herpes simplex virus 1 (HSV-1) (Cooper and Nemerow, 1983Cooper N.R. Nemerow G.R. Complement, viruses, and virus-infected cells.Springer Semin. Immunopathol. 1983; 6: 327-347Crossref PubMed Scopus (38) Google Scholar). However, a recent study found that Factor X was required to promote adenovirus infection in C4-competent as well as C3-deficient mice, suggesting the presence of an unknown C4-dependent mechanism that blocks infection of non-enveloped viruses (Xu et al., 2013Xu Z. Qiu Q. Tian J. Smith J.S. Conenello G.M. Morita T. Byrnes A.P. Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat. Med. 2013; 19: 452-457Crossref PubMed Scopus (126) Google Scholar). Here, we describe a C4-dependent antiviral mechanism that is independent of all downstream complement components. We show that C4 deposition inactivates the capsid of the model non-enveloped virus human adenovirus 5 (Ad5) by interfering with the key capsid disassembly processes of fiber shedding and protein VI exposure, preventing it from entering the cell cytosol and thus blocking infection. To determine whether there are undescribed antibody-dependent antiviral mechanisms, we carried out adenovirus infection experiments in which we ablated known antibody receptor interactions. Using a recombinant mouse-human chimeric monoclonal antibody (mAb) against the main coat protein and primary immunogen of Ad5 (hexon), called 9C12 (9C12-WT), we introduced mutations L234A/L235A (LALA) to prevent Fc gamma receptor (FcγR) binding (Wines et al., 2000Wines B.D. Powell M.S. Parren P.W. Barnes N. Hogarth P.M. The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A.J. Immunol. 2000; 164: 5313-5318Crossref PubMed Scopus (112) Google Scholar) and P329A to prevent C1q interaction (Idusogie et al., 2000Idusogie E.E. Presta L.G. Gazzano-Santoro H. Totpal K. Wong P.Y. Ultsch M. Meng Y.G. Mulkerrin M.G. Mapping of the C1q binding site on Rituxan, a chimeric antibody with a human IgG1 Fc.J. Immunol. 2000; 164: 4178-4184Crossref PubMed Scopus (356) Google Scholar). We performed matched antibody titrations during adenoviral challenge of either wild-type (WT) cells or cells deficient of the intracellular antibody receptor TRIM21 (Figure S1A). TRIM21 detects antibody-bound viruses that have entered the cytosol by binding the IgG Fc region with its PRYSPRY domain (Mallery et al., 2010Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. USA. 2010; 107: 19985-19990Crossref PubMed Scopus (331) Google Scholar). It then becomes activated and undergoes autoubiquitination, which results in recruitment of the proteasome and degradation of the viral particle (Fletcher et al., 2014Fletcher A.J. Mallery D.L. Watkinson R.E. Dickson C.F. James L.C. Sequential ubiquitination and deubiquitination enzymes synchronise the dual sensor and effector functions of TRIM21.Proc. Natl. Acad. Sci. USA. 2014; 112: 10014-10019Crossref Scopus (79) Google Scholar). It is an important component of antibody-mediated protection against viruses such as Ad5 (Bottermann et al., 2018Bottermann M. Foss S. van Tienen L.M. Vaysburd M. Cruickshank J. O’Connell K. Clark J. Mayes K. Higginson K. Hirst J.C. et al.TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.Proc. Natl. Acad. Sci. USA. 2018; 115: 10440-10445Crossref PubMed Scopus (36) Google Scholar). Interestingly, while both anti-Ad5 antibody mutants only minimally affected the persistent fraction of infected WT cells, they strongly reduced neutralization in TRIM21 knockout (KO) cells, P329A more notably so than LALA (Figure 1A, left). TRIM21-independent neutralization was especially prominent at a high antibody concentration (Figure 1A, middle), and use of the P329A mutant largely converted this neutralization from a TRIM21-independent effect to a TRIM21-dependent effect (Figure 1A, right). 293Ts and HeLas do not express FcγRs (Figures S1B–S1D); however, the LALA mutation also affects binding to C1q (Wang et al., 2018Wang X. Mathieu M. Brezski R.J. IgG Fc engineering to modulate antibody effector functions.Protein Cell. 2018; 9: 63-73Crossref PubMed Scopus (171) Google Scholar), suggesting that this mutant may be ablating neutralization because of reduced C1q recruitment, similar to P329A (Figure S1E). To confirm that C1q mediates neutralization of adenovirus, we carried out experiments in TRIM21 KO cells, which allowed us to exclusively assess TRIM21-independent neutralization. Crucially, we observed little neutralization using either buffer (HBS++) or C1q-depleted serum (Figure 1B). However, reconstitution of C1q-depleted serum with C1q protein resulted in potent Ad5 neutralization with the WT antibody, while the LALA and P329A mutants were largely inactive, thereby underlining a role for C1q in Ad5 infection. We investigated the threshold for antibody activation of C1q by determining how many antibody molecules per virus are necessary for efficient C1q-dependent neutralization. To this end, we performed a neutralization assay with normal human serum (NHS) using different ratios of WT:P329A while keeping the total antibody concentration >160 nM (Figure 1C). Using this approach, we determined the EC50 of the WT/P329A ratio to be 0.14 (SEM ± 0.01). Since we previously demonstrated that at saturating concentration (>100 nM) approximately 205 9C12 molecules bind to each Ad5 virion (McEwan et al., 2012McEwan W.A. Hauler F. Williams C.R. Bidgood S.R. Mallery D.L. Crowther R.A. James L.C. Regulation of virus neutralization and the persistent fraction by TRIM21.J. Virol. 2012; 86: 8482-8491Crossref PubMed Scopus (67) Google Scholar), we established the number of WT antibodies per virus at the EC50 as 25.1 (SEM ± 1.7). Notably, however, persistent fraction is only reached when WT outcompetes P329A 10-fold, which corresponds to approximately 185 WT antibodies per virus. This result demonstrates that complement-mediated neutralization occurs at antibody-binding levels well below the maximum occupancy but higher than those required by TRIM21, which can neutralize infection 10-fold with as little as 5 antibodies per virus (McEwan et al., 2012McEwan W.A. Hauler F. Williams C.R. Bidgood S.R. Mallery D.L. Crowther R.A. James L.C. Regulation of virus neutralization and the persistent fraction by TRIM21.J. Virol. 2012; 86: 8482-8491Crossref PubMed Scopus (67) Google Scholar). Having established that C1q mediates Ad5 neutralization in the presence of the mAb 9C12-WT, we tested whether this also occurs with serum IgG. Since we have shown that a threshold of mAb 9C12 coating is required for efficient C1q-dependent neutralization, we tested whether this degree of antibody coating can be achieved by polyclonal serum. First, we determined that the concentration of Ad5 specific antibodies in NHS is 97.2 μg/mL (Figure S1F), which is significantly higher than the concentration of 9C12 required for efficient C1q-dependent neutralization. We then compared binding of polyclonal serum antibody and binding of monoclonal 9C12 to Ad5 at matched Ad5-specific antibody concentrations and found that polyclonal antibodies coated the virus as efficiently as 9C12 (Figure S1G). Next, we performed a time course and incubated virus with either NHS or C1q-depleted serum (Figure 1D). We observed increasing viral neutralization over time using NHS, but not C1q-depleted serum, an effect that was strictly dependent on the presence of IgG in the serum (Figure 1E). Given that C1q is able to neutralize Ad5 infection in the presence of both mAb and polyclonal serum, we sought to determine whether other members of the complement cascade are required. We found that while 9C12 neutralized Ad5 infection in C2- and C3-depleted sera as efficiently as in complete NHS, it was unable to do so in C1q- and C4-depleted sera (Figure 1F), indicating that C4 is required in addition to C1q. To further confirm that the observed Ad5 neutralization was solely dependent on C1 and C4, we attempted to reconstitute activity using only the purified proteins (Figure 1G). Keeping the concentration of C1 constant, we performed a titration of C4 and observed dose-dependent neutralization of Ad5. The EC50 of C4-mediated neutralization was determined as 45.5 μg/mL (SEM ± 3.0), a concentration of C4 ∼10-fold lower than normal serum levels and therefore well within the physiological range. Importantly, neutralization depended on the presence of an intact C1 complex (Figure S2A) and interaction of C1q with antibody (Figure S2B), indicating the activation of the classical pathway. Classical pathway initiation relies on the interaction of C1q with antibody to activate the C1-associated proteases (Figure S2C), which, in the first step of the complement cascade, convert C4 into C4a and the highly reactive thioester C4b (Figure S2D). To directly test for complement activation, we incubated virus and antibody with NHS and monitored the generation of C4b over time (Figure 2A). We observed cleavage of C4 after only 1 min of incubation, and after 15 min, a large proportion of the C4 present in the serum was converted into C4b. This conversion was crucially dependent on an intact complement cascade, as we did not observe cleavage of C4 in C1q-depleted serum (Figure 2A). Activation of the complement cascade and generation of the C4b and C4a fragments yields two hypotheses of how Ad5 neutralization could occur: either liberation of the C4a fragment could act on target cells and render them less permissive to Ad5 infection or generation of the highly reactive thioester C4b results in C4b deposition on the virus, impacting its ability to productively infect. To test which scenario is more likely, we made use of the fact that Ad5 can be engineered to express different transgenes (Figure 2B). Ad5-mCherry was incubated with antibody and NHS for 30 min to allow for complement activation and C4a generation as well as C4b deposition, while Ad5-GFP was kept under control conditions. Ad5-mCherry was added to cells, followed by Ad5-GFP. If neutralization were caused by an effect on the target cells, we would expect equal neutralization of both Ad5-mCherry and Ad5-GFP. However, if neutralization were caused by a direct modification of the viral capsid through C4b deposition, we would only expect neutralization of Ad5-mCherry. This latter outcome is what we observed; Ad5-mCherry was neutralized strongly in a C1q-dependent manner, while Ad5-GFP infection was unaffected (Figure 2B), indicating that neutralization requires direct modification of the viral capsid. C4b contains a highly reactive thioester that is known to covalently attach to any nearby proteins. Consistent with this property, we observed C4-specific high-molecular-weight modifications upon activation of the complement cascade (Figure 2C). These started to appear after 15 min of incubation (Figure S2E) and likely represent C4b attachment to surrounding serum proteins. To test whether C4b also attaches to the Ad5 capsid, we incubated virus and 9C12-WT with NHS and pelleted the complex over a sucrose gradient to remove any proteins not associated with the viral capsid (Figure 2D). While no C4 was pelleted if NHS alone was loaded onto the gradient, we observed a strong C4 signal when virus and antibody were incubated with NHS, indicating that C4b was indeed deposited on the capsid. Again, this was dependent on an intact complement cascade since no C4 was pelleted if C1q- or C4-depleted serum was used. Similarly, we observed a strong signal for C4 if virus and antibody were incubated with C1 and C4, but not if they were incubated with C4 alone (Figure 2D). Taken together, these data suggest that C4b covalently attaches to the viral capsid and mediates neutralization of Ad5. Next, we investigated the mechanism by which C4b deposition on the Ad5 capsid interferes with infection. We first tested whether C4b deposition could interfere with binding to host cell receptors and quantified the number of cell-associated viruses by qPCR (Figure 3A). Interestingly, we did not observe reduced attachment in the presence of C1 and C1/C4; instead, more viral copies were detected in the presence of complement. The same result was obtained when quantifying viral particles by western blot (Figure 3B) and flow cytometry (Figure 3C). Since a known neutralization mechanism by complement is the aggregation of viral particles (Oldstone et al., 1974Oldstone M.B. Cooper N.R. Larson D.L. Formation and biologic role of polyoma virus-antibody complexes. A critical role for complement.J. Exp. Med. 1974; 140: 549-565Crossref PubMed Scopus (35) Google Scholar), we used nanoparticle tracking analysis to demonstrate that C1q does not aid the formation of immune complexes (Figure S3A), which could have explained the increased attachment. Depletion of the main Ad5 receptor coxsackie-and-adenovirus receptor (CAR), however, revealed that in the presence of C1, virus attachment became less dependent on CAR (Figure S3B). This indicates that C1 may mediate binding to another surface receptor, which is consistent with the expression of C1q-receptor and binding proteins at the cell surface. An attractive candidate might be gC1qBP, whose expression is not restricted to phagocytes but has been shown to be expressed on the plasma membrane of various cell types (Soltys et al., 2000Soltys B.J. Kang D. Gupta R.S. Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues.Histochem. Cell Biol. 2000; 114: 245-255Crossref PubMed Scopus (116) Google Scholar). Next, we hypothesized that complement might interfere with the internalization process of viral particles. To test this, we performed a time course to track viral entry. Cells were washed 30 min post Ad5 infection, harvested in 30 min intervals, and stained for surface-bound Ad5 particles (Figures 3D and S3C). While no surface-bound virions could be detected by flow cytometry 90 min post infection, all viral genomes that were associated with the cells at 30 min post infection were still associated with the cells at 90 min post infection (Figure 3D), indicating that the virions were internalized rather than shed into the medium. Host-cell-receptor interaction is crucial for productive Ad5 infection. Ad5 engages two cell surface receptors to trigger entry: it interacts with CAR (Bergelson et al., 1997Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2614) Google Scholar) through its trimeric fiber protein while engaging αvβ3/5 integrins through an RGD motif in its penton base (Bai et al., 1994Bai M. Campisi L. Freimuth P. Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.J. Virol. 1994; 68: 5925-5932PubMed Google Scholar, Wickham et al., 1993Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment.Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1951) Google Scholar). While CAR undergoes a drifting motion on the plasma membrane, the integrin remains static, thereby promoting the release of fiber and penton base from the Ad5 capsid. This results in the release of the membrane-lytic protein VI, which is essential for endosomal lysis and entry of Ad5 into the cytosol (Luisoni et al., 2015Luisoni S. Suomalainen M. Boucke K. Tanner L.B. Wenk M.R. Guan X.L. Grzybek M. Coskun Ü. Greber U.F. Co-option of membrane wounding enables virus penetration into cells.Cell Host Microbe. 2015; 18: 75-85Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Burckhardt et al., 2011Burckhardt C.J. Suomalainen M. Schoenenberger P. Boucke K. Hemmi S. Greber U.F. Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and membrane lytic protein exposure.Cell Host Microbe. 2011; 10: 105-117Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, Meier et al., 2002Meier O. Boucke K. Hammer S.V. Keller S. Stidwill R.P. Hemmi S. Greber U.F. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake.J. Cell Biol. 2002; 158: 1119-1131Crossref PubMed Scopus (384) Google Scholar, Maier et al., 2012Maier O. Marvin S.A. Wodrich H. Campbell E.M. Wiethoff C.M. Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape.J. Virol. 2012; 86: 10821-10828Crossref PubMed Scopus (72) Google Scholar, Wiethoff et al., 2005Wiethoff C.M. Wodrich H. Gerace L. Nemerow G.R. Adenovirus protein VI mediates membrane disruption following capsid disassembly.J. Virol. 2005; 79: 1992-2000Crossref PubMed Scopus (328) Google Scholar). To test whether C4b deposition on the virus interferes with this capsid disassembly process and thus membrane lysis, we analyzed the three processes that need to occur: fiber shedding, protein VI exposure, and membrane lysis. To examine fiber shedding in the presence of deposited C4b, we exploited the fact that Ad5 sheds its fiber not only in response to mechanical cues but also in response to heat (Smith and Nemerow, 2008Smith J.G. Nemerow G.R. Mechanism of adenovirus neutralization by human alpha-defensins.Cell Host Microbe. 2008; 3: 11-19Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, Wiethoff et al., 2005Wiethoff C.M. Wodrich H. Gerace L. Nemerow G.R. Adenovirus protein VI mediates membrane disruption following capsid disassembly.J. Virol. 2005; 79: 1992-2000Crossref PubMed Scopus (328) Google Scholar). We incubated the Ad5 capsid at different temperatures and then performed immunoprecipitation (IP) with 9C12, which precipitates the intact capsid (Figure 4A). Upon heating Ad5 to 49°C, fiber and penton base were no longer detected by western blot, indicating that they were not associated with the capsid but had been shed. When the same experiment was repeated in the presence of C1 and C4 (Figure 4B), both fiber and penton base were still associated with the Ad5 capsid when it was heated to 49°C, indicating that C4b deposition on the viral capsid does interfere with Ad5 capsid disassembly. Next, we investigated whether C4 prevents exposure of protein VI during infection by confocal microscopy (Figure 4C), which only detects partially disassembled capsids where the pVI epitope is unmasked (Burckhardt et al., 2011Burckhardt C.J. Suomalainen M. Schoenenberger P. Boucke K. Hemmi S. Greber U.F. Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and membrane lytic protein exposure.Cell Host Microbe. 2011; 10: 105-117Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We observed good protein VI exposure in the presence of 9C12-WT and C1, but the amount of protein VI staining significantly decreased in the presence of C1 and C4, demonstrating that C4b deposition inhibits protein VI exposure. This is consistent with the lack of capsid disassembly indicated by blotting for fiber and penton base. Finally, we tested whether the reduced exposure of membrane-lytic protein VI is sufficient to prevent the virus from penetrating the endosomal membrane. We used Galectin 3 as a marker for endosomal lysis since it is recruited to ruptured endosomes (Aits et al., 2015Aits S. Kricker J. Liu B. Ellegaard A.M. Hämälistö S. Tvingsholm S. Corcelle-Termeau E. Høgh S. Farkas T. Holm Jonassen A. et al.Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.Autophagy. 2015; 11: 1408-1424Crossref PubMed Scopus (193) Google Scholar, Maier et al., 2012Maier O. Marvin S.A. Wodrich H. Campbell E.M. Wiethoff C.M. Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape.J. Virol. 2012; 86: 10821-10828Crossref PubMed Scopus (72) Google Scholar, Luisoni et al., 2016Luisoni S. Bauer M. Prasad V. Boucke K. Papadopoulos C. Meyer H. Hemmi S. Suomalainen M. Greber U.F. Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells.Matters. 2016; : 1-9Google Scholar) and forms distinct puncta proportional to the multiplicity of infection (MOI) of Ad5 used (Figure S4). Similarly to the protein VI staining microscopy, we observed nice puncta formation in response to Ad5 infection in the presence of 9C12-WT and C1; however, significantly less Gal3 puncta were observed during infection in the presence of C1 and C4 (Figure 4D). This suggests that in the presence of complement, Ad5 penetrates endosomes less efficiently. To directly demonstrate that viruses are trapped in endosomes in the presence of complement and prevented from entering the cytosol, we used a variation of the membrane penetration assay described by Suomalainen et al., 2013Suomalainen M. Luisoni S. Boucke K. Bianchi S. Engel D.A. Greber U.F. A direct and versatile assay measuring membrane penetration of adenovirus in single cells.J. Virol. 2013; 87: 12367-12379Crossref PubMed Scopus (55) Google Scholar. We electroporated HeLa cells with an anti-Fab-FITC antibody and infected them with Ad5 in the presence of 9C12-WT and complement. Any viruses that have penetrated the endosome and escaped into the cytosol will be bound by the electroporated anti-Fab antibody. Cells were then fixed, permeabilized, and stained with an anti-Fab followed by an AF647-labeled secondary antibody. Therefore, all cytosolic viruses will acquire a dual FITC and AF647 signal, while endosomal viruses will only be labeled with the AF647 signal. Investigating whether 9C12 alone alters egress into the cytosol, we found that around 20% of viruses were cytosolic regardless of the presence of 9C12 (Figure 5A, middle). Upon the addition of complement, however, the percentage of cytosolic viruses dropped to less than 2% (Figure 5A, left and right), further corroborating our data that C1 and C4 specifically interfere with virus entry into the cytosol. Once Ad5 has escaped the endosome, hexon recruits dynein and travels along microtubules to the nucleus, where it interacts with the nuclear pore complex to deliver the viral genome into the nucleus (Bremner et al., 2009Bremner K.H. Scherer J. Yi J. Vershinin M. Gross S.P. Vallee R.B. Adenovirus transport via direct interaction of cytoplasmic dynein with the viral capsid Hexon subunit.Cell Host Microbe. 2009; 6: 523-535Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Leopold et al., 2000Leopold P.L. Kreitzer G. Miyazawa N. Rempel S. Pfister K.K. Rodriguez-Boulan E. Crystal R.G. Dynein- and microtubule-mediated translocation of adenovirus Serotype 5 occurs after endosomal lysis.Hum. Gene Ther. 2000; 11: 151-165Crossref PubMed Scopus (215) Google Scholar, Suomalainen et al., 1999Suomalainen M. Nakano M.Y. Keller S. Boucke K. Stidwill R.P. Greber U.F. Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.J. Cell Biol. 1999; 144: 657-672Crossref PubMed Scopus (380) Google Scholar, Cassany et al., 2015Cassany A. Ragues J. Guan T. Bégu D. Wodrich H. Kann M. Nemerow G.R. Gerace L. Nuclear import of adenovirus DNA involves direct interaction of Hexon with an N-terminal domain of the nucleoporin Nup214.J. Virol. 2015; 89: 1719-1730Crossref PubMed Scopus (48) Google Scholar, Trotman et al., 2001Trotman L.C. Mosberger N. Fornerod M. Stidwill R.P. Greber U.F. Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1.Nat. Cell Biol. 2001; 3: 1092-1100Crossref PubMed Scopus (255) Google Scholar). Given" @default.
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• W2925325810 title "Complement C4 Prevents Viral Infection through Capsid Inactivation" @default.
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• W2925325810 doi "https://doi.org/10.1016/j.chom.2019.02.016" @default.
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