2.5 Conclusion
3.3.7 Less S protein is incorporated into virions after NOC treatment of
ST cells infected with TGEV were treated with DMSO or NOC. After cell lysis as well as virus particle concentration by ultracentrifugation the incorporation of S into virions was examined by SDS-PAGE followed by Western blot (Fig. 3-9). Mock-infected cells served as negative control. In infected ST cells, not treated with NOC, S protein was detected in virus particles after ultracentrifugation. In cells treated with NOC a very weak or nearly no signal was observed for the S protein incorporated into viral particles. The signal for TGEV S, M and N proteins were similar in the corresponding cell lysates and comparable amounts of M and N protein were detectable in
96
concentrated virions irrespective of a Nocodazole treatment. This indicates a reduced S incorporation into virus particles due to depolymerization of tubulins by NOC treatment of infected ST cells.
Fig. 3-9 TGEV particle assay 24 hpi.
Detection of viral spike (S), nucleocapsid (N), and membrane (M) proteins in cell lysates and pelleted supernatants of TGEV- or mock-infected ST cells. Cells were either treated with NOC, DMSO or medium. Virus particles were concentrated by ultracentrifugation.
3.4 Discussion
Viruses rely on the host cell machinery for successful replication (SAKAGUCHI et al.
1996; KONAN et al. 2003; CHOE et al. 2005; BELOV et al. 2007; BESKE et al. 2007;
MOFFAT et al. 2007; OOSTRA et al. 2007). Nevertheless, more detailed knowledge on virus-host interaction would raise therapeutic tools for infection control. CoVs use the host secretory pathway during their replication cycle. The vesicular transport on secretory pathways is mostly mediated by microtubules and the corresponding motor proteins (FOKIN et al. 2014). A strong effect on RNA replication was observed when ER-to-Golgi transport was impaired. In the case of MHV it was shown that the early secretory pathway is important during the formation of the replication complex (OOSTRA et al. 2007; VERHEIJE et al. 2008; KNOOPS et al. 2010). However, the incorporation of CoV S proteins into virus particles is required for the release of infectious progeny. Information about cellular factors interacting with coronaviral S proteins would be important to understand the virus infection process.
97
In this study we searched for interaction partners of the last 39 aa stretches of TGEV, HCoV NL63 and 229E S cytoplasmic domains (all containing a tyrosine-based motif).
Four different tubulin alpha and beta chains (TUBB2A, TUBB4A, TUBB6 and TUBA4A) were detected to interact with TGEV-S and HCoV-229E-S. Regarding HCoV NL63 only an interaction for TUBB2A was noticed. Either NL63-S-39aa-GFP-NT does not bind certain tubulins or just with low affinity. A destroyed or impaired binding of NL63-S to some tubulins due to the GFP-tag could be possible as well.
For many viruses a close association with cytoskeletal elements was shown (LUFTIG 1982). Adenovirus type 2 and 5 particles as well as reovirus particles interact with
Moreover, tubulin was already found packaged into virions of Epstein-Barr virus, human cytomegalovirus, and murine leukemia virus (WANG et al. 2003;
JOHANNSEN et al. 2004; VARNUM et al. 2004). By confocal microscopy, we observed a partial co-localization of the full length TGEV S protein with the authentic β-tubulin in ST cells. However, some S proteins seemed to lay on the tubulin filaments suggesting an indirect interaction via motor proteins like dynein or kinesin.
For several viruses like adenovirus, African swine fever virus, canine parvovirus, herpes simplex virus as well as lyssavirus and rabies virus an interaction either of the whole virus capsid or of single viral proteins with cytoplasmic dynein was examined (JACOB et al. 2000; RAUX et al. 2000; YE et al. 2000; ALONSO et al. 2001;
SUIKKANEN et al. 2003; DOUGLAS et al. 2004; KELKAR et al. 2004). Viruses may have evolved microtubule-binding motifs or similar amino acid sequences complementary to motifs in dynein for successful interaction (PASICK et al. 1994;
LEOPOLD & PFISTER 2006).
To further examine the interaction of the Alphacoronavirus S proteins with tubulin, nocodazole (NOC) – a drug inducing microtubule depolymerization – was used.
Additionally, the importance of the tyrosine-based motif within the cytoplasmic domain of the Alphacoronavirus S proteins of interest was examined. Therefore, a TGEV S mutant with a destroyed tyrosine-based retention signal (TGEV S Y/A) was used as well as a representative of the genus Betacoronavirus (SARS-CoV S), which
98
contains no tyrosine motif. ST cells transfected with cDNA encoding the last 39 amino acid stretches of TGEV, NL63, 229E and SARS-CoV S cytoplasmic domain or with the TGEV S and TGEV S Y/A full length cDNA were treated with DMSO or NOC.
To analyze CoV S protein localization during early assembly events, cells were fixed 7 hpi (RISCO et al. 1998; VOGELS et al. 2011). Mock treated cells showed an accumulation of the S protein near the nucleus whereas NOC treated cells showed a scattered pattern within the cytoplasm. All tested constructs showed similar expression and distribution pattern either in DMSO or in NOC treated cells. Thus, the tyrosine-based signal is not essential for the association with β-tubulins, whereby the charge-rich region in general is important. Moreover, the cytoplasmic domain of Betacoronavirus S proteins (like in this case SARS-CoV S) is equally involved in tubulin interaction. In TGEV-infected ST cells similar results were obtained 7 hpi.
Also here, instead of an accumulation of S proteins they were distributed in the cytosol after NOC treatment. CoV assembly occurs at the ERGIC. Thus, S proteins accumulate near the nucleus in DMSO treated cells 7 hpi. A redistribution in NOC treated cells was already shown for different viral proteins, like Crimean-Congo hemorrhagic fever virus nucleocapsid protein (SIMON et al. 2009) or herpes simplex virus 1 capsids (SODEIK et al. 1997). In the two tested chiropteran cells lines (HypNi/1.1 and PipNi/1) similar results as for S-transfected or TGEV-infected ST cells were obtained, suggesting a conserved viral strategy to use the host for its own advantage during replication. Due to the assumption that many human and animal CoVs originated from bats and that most eukaryotic cells contain microtubules, a conserved microtubule-dependent CoV replication strategy is likely.
Furthermore, we demonstrated that NOC leads to a reduced amount of released infectious virus particles. Less virus yield after cytoskeleton disruption was already shown for moloney murine leukaemia virus and vaccinia virus (PAYNE &
KRISTENSSON 1982; SATAKE & LUFTIG 1982). To find out which step during The TGEV replication cycle is mostly influenced by the interaction of S with tubulin, cells were drug-treated at different time points (before, while, or after infection). A significantly lower virus titer in the cell culture supernatant of NOC treated cells compared to untreated cells was detected, whereas no differences in viral titers concerning the various time points could be observed. CoVs uses the host secretory pathway to be transported from ER-to-Golgi apparatus as well as during virus assembly (OOSTRA et al. 2007; VERHEIJE et al. 2008; KNOOPS et al. 2010;
99
VOGELS et al. 2011). An impaired transport of viral components and an affected assembly may explain the decrease in virus yield. For MHV, microtubules are important for neuronal transport, replication and viral spread (BISWAS & DAS SARMA 2014). Also here, a reduced efficiency of viral infection was observed by using microtubule disrupting drugs (BISWAS & DAS SARMA 2014). TGEV was able to enter the cells in the absence or presence of NOC, it was able to replicate and to egress but the infection was less efficient regarding the amount of newly formed infectious virions. Due to the fact that depolymerized microtubules did not inhibit TGEV infection completely, virus components have to use other pathways for trafficking beside tubulin filaments to finish their replication cycle. Examinations on adeno-associated, herpes and vaccinia virus showed no complete blocking of infection after microtubule disruption as well (SODEIK et al. 1997; PLOUBIDOU et al.
2000; SANLIOGLU et al. 2000). Also here, an alternative, microtubule-independent pathway used by the virus was suggested (XIAO & SAMULSKI 2012). An actin-dependent strategy for virus movement could be possible (CUDMORE et al. 1997;
LANIER & VOLKMAN 1998; SANLIOGLU et al. 2000). For paramyxoviruses like measles virus actin seems to be involved in protein movement towards the plasma membrane as well as the budding process (EHRNST & SUNDQVIST 1975;
STALLCUP et al. 1983; BOHN et al. 1986). Regarding vaccinia virus, microfilaments affect intracellular movement as well as virus release (STOKES 1976).
Nucleocapsids of a nucleopolyhedrovirus utilize actin cables for their transport to and/ or into the nucleus (LANIER & VOLKMAN 1998). However, lower virus titers may correlate with the differentially distributed S protein in NOC treated cells, too.
Actually, S accumulates near the ERGIC where it interacts with the M protein to be incorporated into newly assembled virions (OPSTELTEN et al. 1995; NGUYEN &
HOGUE 1997). When microtubules are depolymerized S proteins as well as the ERGIC and Golgi compartment are scattered throughout the cell. Due to the diffuse distribution of S and the two compartments which are important for successful TGEV assembly less interaction of viral proteins during assembly may be possible. An impaired transport of virus particles from the Golgi compartment to the plasma membrane may also be a reason for lower viral titers. Concerning this matter, a less organized cytoplasm with dispersed Golgi stacks in NOC treated ST cells was already shown by Risco et al. 1998. Additionally, TGEV virions were seen in these disrupted Golgi stacks whereas only a few were detected on extracellular surfaces
100
(RISCO et al. 1998). A reason may be a microtubule-dependent transport of vesicles between the Golgi compartment and the plasma membrane (RISCO et al. 1998). To find out if S incorporation into virions is influenced by NOC treatment, virus particles were concentrated out of the supernatant of NOC-treated infected cells and compared to mock-treated cells. In this case, the amount of S protein was similar in DMSO and NOC treated cell lysates, whereas S protein quantity was reduced in pelleted virus particles when infected cells were treated with NOC. This fact fits to the calculated smaller amount of released infectious virus particles measured in drug treated cells.
3.5 Conclusion
Our results show that Alpha- and Betacoronavirus S proteins interact with their charge-rich region of their cytoplasmic domains with tubulin beta chains, regardless if a tyrosine-based signal is present or not. An interaction with microtubules facilitates TGEV replication and infection efficiency but the depolymerization of microtubules did not inhibit it completely. Thus, an interaction of S with tubulin may help the virus during replication, assembly as well as the budding process and supports viral particle transport to the plasma membrane.
Authors’ contributions
ATR, AVB and CSW conceived and designed the study. GP and JM performed the mass spectrometry analysis. PM and YML accomplished Invitrogen/ Life Technologies Gateway cloning and GFP Trap® pull down assay. ATR performed immunofluorescence studies and plaque assays. Sandra Bauer performed virus particle assay. ATR, PM, YML, GP, JM, AVB, and CSW analyzed the data. ATR and CSW drafted the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors have declared that no competing interests exist.
101 Funding
Financial support was provided by a grant to CSW (SCHW 1408/1.1) from the German Research Foundation (DFG) and by the “Bundesministerium fuer Bildung und Forschung” of the German Government (Zoonosis Network, Consortium on ecology and pathogenesis of SARS, project code 01KI1005A,F to AvB.
CSW is funded by the Emmy Noether Programme from the DFG. ATR is a recipient of a Georg-Christoph-Lichtenberg PhD fellowship from the Ministry for Science and Culture of Lower Saxony.
Acknowledgements
This work was performed by ATR in partial fulfillment of the requirements for a Dr.
rer. nat. degree from the University of Veterinary Medicine Hannover.
We are grateful to L. Enjuanes, E. Snijder, E. Kremmer and H.-P. Hauri for providing antibodies, plasmids, and virus. We thank M. Müller and C. Drosten for the chiropteran cell lines (HypNi/1.1, PipNi/1). Thanks to S. Bauer for technical assistance.
3.6 References
ALONSO, C., J. MISKIN, B. HERNAEZ, P. FERNANDEZ-ZAPATERO, L. SOTO, C. CANTO, I.
RODRIGUEZ-CRESPO, L. DIXON & J. M. ESCRIBANO (2001): African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.
J Virol 75, 9819-9827
BABISS, L. E., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING, U. R. RAY & B. N. FIELDS (1979): Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J Virol 30, 863-874
BELOV, G. A., N. ALTAN-BONNET, G. KOVTUNOVYCH, C. L. JACKSON, J. LIPPINCOTT-SCHWARTZ & E. EHRENFELD (2007): Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. J Virol 81, 558-567
BEN-ZE'EV, A., M. HOROWITZ, H. SKOLNIK, R. ABULAFIA, O. LAUB & Y. ALONI (1981): The metabolism of SV40 RNA is associated with the cytoskeletal framework. Virology 111, 475-487 BESKE, O., M. REICHELT, M. P. TAYLOR, K. KIRKEGAARD & R. ANDINO (2007): Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex. J Cell Sci 120, 3207-3218
BISWAS, K. & J. DAS SARMA (2014): Effect of microtubule disruption on neuronal spread and replication of demyelinating and nondemyelinating strains of mouse hepatitis virus in vitro. J Virol 88, 3043-3047
BOHN, W., G. RUTTER, H. HOHENBERG, K. MANNWEILER & P. NOBIS (1986): Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149, 91-106
102
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318 CALISHER, C. H., J. E. CHILDS, H. E. FIELD, K. V. HOLMES & T. SCHOUNTZ (2006): Bats:
important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531-545
CHINESE, S. M. E. C. (2004): Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 303, 1666-1669
CHOE, S. S., D. A. DODD & K. KIRKEGAARD (2005): Inhibition of cellular protein secretion by picornaviral 3A proteins. Virology 337, 18-29
CUDMORE, S., I. RECKMANN & M. WAY (1997): Viral manipulations of the actin cytoskeleton.
Trends in microbiology 5, 142-148
DEWERCHIN, H. L., L. M. DESMARETS, Y. NOPPE & H. J. NAUWYNCK (2014): Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes. Vet Res 45, 17
DOUGLAS, M. W., R. J. DIEFENBACH, F. L. HOMA, M. MIRANDA-SAKSENA, F. J. RIXON, V.
VITTONE, K. BYTH & A. L. CUNNINGHAM (2004): Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport. J Biol Chem 279, 28522-28530
EHRNST, A. & K. G. SUNDQVIST (1975): Polar appearance and nonligand induced spreading of measles virus hemagglutinin at the surface of chronically infected cells. Cell 5, 351-359
FOKIN, A. I., I. B. BRODSKY, A. V. BURAKOV & E. S. NADEZHDINA (2014): Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors.
Biochemistry. Biokhimiia 79, 879-893
GE, X. Y., J. L. LI, X. L. YANG, A. A. CHMURA, G. ZHU, J. H. EPSTEIN, J. K. MAZET, B. HU, W.
ZHANG, C. PENG, Y. J. ZHANG, C. M. LUO, B. TAN, N. WANG, Y. ZHU, G. CRAMERI, S. Y.
ZHANG, L. F. WANG, P. DASZAK & Z. L. SHI (2013): Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538
GODEKE, G. J., C. A. DE HAAN, J. W. ROSSEN, H. VENNEMA & P. J. ROTTIER (2000): Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein. J Virol 74, 1566-1571
GRAHAM, R. L. & R. S. BARIC (2010): Recombination, reservoirs, and the modular spike:
mechanisms of coronavirus cross-species transmission. J Virol 84, 3134-3146
GUAN, Y., B. J. ZHENG, Y. Q. HE, X. L. LIU, Z. X. ZHUANG, C. L. CHEUNG, S. W. LUO, P. H. LI, L.
J. ZHANG, Y. J. GUAN, K. M. BUTT, K. L. WONG, K. W. CHAN, W. LIM, K. F. SHORTRIDGE, K. Y.
YUEN, J. S. PEIRIS & L. L. POON (2003): Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276-278
GUPTA, S., B. P. DE, J. A. DRAZBA & A. K. BANERJEE (1998): Involvement of actin microfilaments in the replication of human parainfluenza virus type 3. J Virol 72, 2655-2662 HAN, X., Z. LI, H. CHEN, H. WANG, L. MEI, S. WU, T. ZHANG, B. LIU & X. LIN (2012): Influenza virus A/Beijing/501/2009(H1N1) NS1 interacts with beta-tubulin and induces disruption of the microtubule network and apoptosis on A549 cells. PloS one 7, e48340
HARA, Y., R. HASEBE, Y. SUNDEN, K. OCHIAI, E. HONDA, Y. SAKODA & T. UMEMURA (2009):
Propagation of swine hemagglutinating encephalomyelitis virus and pseudorabies virus in
103
dorsal root ganglia cells. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 71, 595-601
HEALD, R. & E. NOGALES (2002): Microtubule dynamics. Journal of cell science 115, 3-4
HENRY SUM, M. S. (2015): The involvement of microtubules and actin during the infection of Japanese encephalitis virus in neuroblastoma cell line, IMR32. Biomed Res Int 2015, 695283 HSIEH, M. J., P. J. WHITE & C. W. POUTON (2010): Interaction of viruses with host cell molecular motors. Current opinion in biotechnology 21, 633-639
HYDE, J. L., L. K. GILLESPIE & J. M. MACKENZIE (2012): Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center. Journal of virology 86, 4110-4122
JACOB, Y., H. BADRANE, P. E. CECCALDI & N. TORDO (2000): Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. J Virol 74, 10217-10222
JOHANNSEN, E., M. LUFTIG, M. R. CHASE, S. WEICKSEL, E. CAHIR-MCFARLAND, D. ILLANES, D. SARRACINO & E. KIEFF (2004): Proteins of purified Epstein-Barr virus. Proceedings of the National Academy of Sciences of the United States of America 101, 16286-16291
KELKAR, S. A., K. K. PFISTER, R. G. CRYSTAL & P. L. LEOPOLD (2004): Cytoplasmic dynein mediates adenovirus binding to microtubules. Journal of virology 78, 10122-10132
KNOOPS, K., C. SWETT-TAPIA, S. H. VAN DEN WORM, A. J. TE VELTHUIS, A. J. KOSTER, A. M.
MOMMAAS, E. J. SNIJDER & M. KIKKERT (2010): Integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus RNA synthesis and virus-induced remodeling of endoplasmic reticulum membranes. Journal of virology 84, 833-846
KONAN, K. V., T. H. GIDDINGS, JR., M. IKEDA, K. LI, S. M. LEMON & K. KIRKEGAARD (2003):
Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J Virol 77, 7843-7855
KRIJNSE-LOCKER, J., M. ERICSSON, P. J. ROTTIER & G. GRIFFITHS (1994): Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55-70
KUHL, A., M. HOFFMANN, M. A. MULLER, V. J. MUNSTER, K. GNIRSS, M. KIENE, T. S. TSEGAYE, G. BEHRENS, G. HERRLER, H. FELDMANN, C. DROSTEN & S. POHLMANN (2011): Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells. J Infect Dis 204 Suppl 3, S840-849
KYHSE-ANDERSEN, J. (1984): Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journal of biochemical and biophysical methods 10, 203-209
LAKADAMYALI, M., M. J. RUST, H. P. BABCOCK & X. ZHUANG (2003): Visualizing infection of individual influenza viruses. Proc Natl Acad Sci U S A 100, 9280-9285
LANIER, L. M. & L. E. VOLKMAN (1998): Actin binding and nucleation by Autographa california M nucleopolyhedrovirus. Virology 243, 167-177
LAU, S. K., P. C. WOO, K. S. LI, Y. HUANG, H. W. TSOI, B. H. WONG, S. S. WONG, S. Y. LEUNG, K. H. CHAN & K. Y. YUEN (2005): Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040-14045
LENK, R. & S. PENMAN (1979): The cytoskeletal framework and poliovirus metabolism. Cell 16, 289 301
104
LEOPOLD, P. L. & K. K. PFISTER (2006): Viral strategies for intracellular trafficking: motors and microtubules. Traffic 7, 516-523
LI, W., Z. SHI, M. YU, W. REN, C. SMITH, J. H. EPSTEIN, H. WANG, G. CRAMERI, Z. HU, H.
ZHANG, J. ZHANG, J. MCEACHERN, H. FIELD, P. DASZAK, B. T. EATON, S. ZHANG & L. F.
WANG (2005): Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679 LIU, C., J. TANG, Y. MA, X. LIANG, Y. YANG, G. PENG, Q. QI, S. JIANG, J. LI, L. DU & F. LI (2015):
Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol 89, 6121-6125 LUBY-PHELPS, K. (2000): Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. International review of cytology 192, 189-221
LUFTIG, R. B. (1982): Does the cytoskeleton play a significant role in animal virus replication? J Theor Biol 99, 173-191
LUFTIG, R. B. & R. R. WEIHING (1975): Adenovirus binds to rat brain microtubules in vitro. J Virol 16, 696-706
MEIER, O., K. BOUCKE, S. V. HAMMER, S. KELLER, R. P. STIDWILL, S. HEMMI & U. F. GREBER (2002): Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. The Journal of cell biology 158, 1119-1131
MILES, B. D., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING & J. WEBER (1980):
Quantitation of the interaction between adenovirus types 2 and 5 and microtubules inside infected cells. Virology 105, 265-269
MIRANDA-SAKSENA, M., P. ARMATI, R. A. BOADLE, D. J. HOLLAND & A. L. CUNNINGHAM (2000): Anterograde transport of herpes simplex virus type 1 in cultured, dissociated human and rat dorsal root ganglion neurons. J Virol 74, 1827-1839
MOFFAT, K., C. KNOX, G. HOWELL, S. J. CLARK, H. YANG, G. J. BELSHAM, M. RYAN & T.
WILEMAN (2007): Inhibition of the secretory pathway by foot-and-mouth disease virus 2BC protein is reproduced by coexpression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C. J Virol 81, 1129-1139
MULLER, M. A., V. S. RAJ, D. MUTH, B. MEYER, S. KALLIES, S. L. SMITS, R. WOLLNY, T. M.
BESTEBROER, S. SPECHT, T. SULIMAN, K. ZIMMERMANN, T. BINGER, I. ECKERLE, M.
TSCHAPKA, A. M. ZAKI, A. D. OSTERHAUS, R. A. FOUCHIER, B. L. HAAGMANS & C. DROSTEN (2012): Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines. mBio 3,
NAKANO, M. Y., K. BOUCKE, M. SUOMALAINEN, R. P. STIDWILL & U. F. GREBER (2000): The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol. J Virol 74, 7085-7095
NGUYEN, V. P. & B. G. HOGUE (1997): Protein interactions during coronavirus assembly. J Virol 71, 9278-9284
NOGALES, E. (2000): Structural insights into microtubule function. Annual review of biochemistry 69, 277-302
OOSTRA, M., E. G. TE LINTELO, M. DEIJS, M. H. VERHEIJE, P. J. ROTTIER & C. A. DE HAAN (2007): Localization and membrane topology of coronavirus nonstructural protein 4:
involvement of the early secretory pathway in replication. J Virol 81, 12323-12336
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):