S and M proteins of MHV interact specifically with each other and form large heterocomplexes post-translationally but with different kinetics. While unmodified M proteins immediately associate with S proteins after synthesis, newly synthesized S proteins are found in these complexes after 10-20 min. S protein folding is necessary for its interaction with M proteins and is the rate-limiting step, whereas under reducing conditions no M-S complexes are formed (OPSTELTEN et al. 1994).
Correct S protein folding involves formation of disulfide bonds, addition of N-linked sugars, as well as its assembly into homo-oligomers, which occurs slowly
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(VENNEMA et al. 1990; OPSTELTEN et al. 1993; OPSTELTEN et al. 1995). In contrast, the final conformation of M proteins is glycosylated but without inter- or intramolecular disulfide bonds (OPSTELTEN et al. 1993). In MHV M and S protein co-expressing cells, heteromultimeric complexes are transported beyond the ERGIC and accumulate at the Golgi apparatus (OPSTELTEN et al. 1995). M proteins themselves are able to form complexes by M-M interactions as well. They accumulate at the Golgi complex in homomultimeric and detergent-insoluble structures (LOCKER et al. 1995). During CoV particle assembly only M and E proteins are required and VLPs are built, whereas S proteins are incorporated when present (BOS et al. 1996; VENNEMA et al. 1996). Interestingly, N proteins are not required for VLP assembly and were not always taken into VLPs when present (VENNEMA et al. 1996). Such nucleocapsid-less particles show a typical CoV morphology but a lower density compared to virions, as this is shown for IBV (MACNAUGHTON & DAVIES 1980). Beside M-S complexes, M proteins are able to interact with HE proteins and form M-HE complexes leading to HE incorporation into virus particles. Thus, M proteins play a crucial role in virus assembly retaining viral glycoproteins at internal membranes (NGUYEN & HOGUE 1997).
However, CoV S proteins show low sequence conservation. Approximately 30 % sequence identity is found in the S2 region responsible for membrane fusion (CAVANAGH 1995). The highly conserved sequence KWPW(Y/W)VWL is present at the transition of the transmembrane and ectodomain, which is probably involved in membrane fusion as well but is dispensable for S incorporation into virus particles (BOSCH et al. 2005). Additionally, in the cytoplasmic domain conserved cysteines (approximately 24 %) are present (BOSCH et al. 2005). Regarding MHV and SARS-CoV S cytoplasmic tail, especially their charge-rich region seems to play a key role during M interaction (YE et al. 2004; BOSCH et al. 2005; MCBRIDE et al. 2007). By the help of a recombinant fMHV mutant, containing a chimeric S gene (ectodomain of FIPV, transmembrane and endodomain of MHV), a host switching event to feline cells was detected due to the FIPV ectodomain. Nonetheless, interaction with MHV M protein was still possible, resulting in S chimera incorporation into virus particles due to the S carboxy-terminus derived from MHV (KUO et al. 2000). In contrast, for TGEV S-M interaction neither the S cytoplasmic domain cysteine-rich motif nor the tyrosine-based retention signal are essential (GELHAUS et al. 2014). All tested TGEV S chimera, where the charge-rich region was exchanged by corresponding
142
human- and bat-derived S sequences, were intracellularly retained closely to M proteins in co-transfected cells. Nevertheless, investigated TGEV S chimera showed different expression pattern when expressed alone. However, all testes S constructs still contained positively or negatively charged amino acids. For further investigations, S mutants harboring only uncharged amino acids downstream their cysteine-rich region as well as the dibasic signal should be examined regarding their importance during S-M protein interaction. Results may give a hint if the charge-rich region and the dibasic signal of the TGEV S protein are necessary at all.
S protein regions important for the interaction with M proteins may depend on the CoV genera. For Betacoronaviruses the S cytoplasmic domain seems to be necessary, whereas for Alphacoronaviruses the S ectodomain is probably involved in M protein association. A reason therefore may lay in S protein modifications during maturation. For example, concerning MHV and SARS-CoV, S protein ectodomains are cleaved into S1 and S2 subunits to reach full functional activity, which is regarding TGEV not tested yet.
5.4 Conclusion
CoVs underlay high mutation and recombination rates during their replication cycle, although they show a narrow host range. Nonetheless, several host shifting events are known for these enveloped RNA viruses including successful animal-to-human transmission. Especially, bats represent high risk factors for zoonotic host shifting.
Therefore, better understanding of CoV life cycle is necessary. In this study, we confirmed that the Alphacoronavirus S protein is the determinant for species restriction due to its function of receptor binding. Virus attachment to the species specific receptor is the first hurdle the virus has to overcome. However, during virus replication, assembly and budding, further cellular factors seem to be involved and play a crucial role in host restriction. We were able to identify tubulins as interaction partners of the CoV S protein cytoplasmic domain. This association highly likely supports the S protein transport during the virus assembly and budding process. Due to the fact that tubulins are conserved among eukaryotic cells this host factor is available also in potential new hosts and does not represent a species barrier.
Moreover, the TGEV S protein tyrosine-based retention signal is not essential for S-M interaction. Here, its ectodomain, the charged amino acids or the dibasic motif
143
within the charge-rich region of the cytoplasmic domain might play a key role.
Sequence differences in the S charge-rich region may not be a barrier for coronaviral evolution, as long as it contains a certain amount of charged amino acids.
Recombination events which may not disturb S-M interaction and incorporation of S proteins into virions might extend the host range of newly evolved CoVs.
Further insights into CoV life cycle, usage of host cell machinery as well as the identification and characterization of additional involved cellular factors may promote antiviral target investigations.
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Appendix
Nucleotide and amino acid sequences
TGEV (PUR-46 MAD) S protein
Nucleotide sequence (4344 bp)
1 atgaaaaaac tatttgtggt tttggtcgta atgccattga tttatggaga 51 caattttcct tgttctaaat tgactaatag aactataggc aaccagtgga 101 atctcattga aaccttcctt ctaaactata gtagtaggtt accacctaat
150
151 Amino acid sequence (1447 aa)
accession no. CAB91145
1 MKKLFVVLVV MPLIYGDNFP CSKLTNRTIG NQWNLIETFL LNYSSRLPPN 51 SDVVLGDYFP TVQPWFNCIR NDSNDLYVTL ENLKALYWDY ATENITWNHR 101 QRLNVVVNGY PYSITVTTTR NFNSAEGAII CICKGSPPTT TTESSLTCNW
1 atgaagattt tgttaatatt agcgtgtgtg attgcatgcg catgtggaga 51 acgctattgt gctatgaaat ccgatacaga tttgtcatgt cgcaatagta 101 cagcgtctga ttgtgagtca tgcttcaacg gaggcgatct tatttggcat
152
1 MKILLILACV IACACGERYC AMKSDTDLSC RNSTASDCES CFNGGDLIWH 51 LANWNFSWSI ILIVFITVLQ YGRPQFSWFV YGIKMLIMWL LWPVVLALTI 101 FNAYSEYQVS RYVMFGFSIA GAIVTFVLWI MYFVRSIQLY RRTKSWWSFN
1 atggccaagg gattctacat ttccaaggcc ctgggcatcc tgggcatcct 51 cctcggcgtg gcggccgtgg ccaccatcat cgctctgtct gtggtgtatg 101 cccaggagaa gaacaagaat gccgagcatg tcccccaagc ccccacgtcg
153
154 Amino acid sequence (963 aa)
accession no. NP_999442
1 MAKGFYISKA LGILGILLGV AAVATIIALS VVYAQEKNKN AEHVPQAPTS 51 PTITTTAAIT LDQSKPWNRY RLPTTLLPDS YFVTLRPYLT PNADGLYIFK 101 GKSIVRLLCQ ESTDVIIIHS KKLNYTTQGH MVVLRGVGDS QVPEIDRTEL 151 VELTEYLVVH LKGSLQPGHM YEMESEFQGE LADDLAGFYR SEYMEGNVKK 201 VLATTQMQST DARKSFPCFD EPAMKATFNI TLIHPNNLTA LSNMPPKGSS 251 TPLAEDPNWS DTEFETTPVM STYLLAYIVS ESQSVNETAQ NGVLIRIWAR 301 PNAIAEGHGM YALNVTGPIL NFFANHYNTS YPLPKSDQIA LPDFNAGAME 351 NWGLVTYREN ALLFDPQSSS ISNKERVVTV IAHELAHQWF GNLVTLAWWN 401 DLWLNEGFAS YVEYLGADHA EPTWNLKDLI VPGDVYRVMA VDALASSHLL 451 TTPAEEVNTP AQISEMFDSI SYSKGASVIR MLSNFLTEDL FKEGLASYLH 501 AFAYQNTTYL DLWEHLQKAV DAQTSIRLPD TVRAIMDRWT LQMGFPVITV 551 DTKTGNISQK HFLLDSESNV TRSSAFDYLW IVPISSIKNG VMQDHYWLRD 601 VSQAQNDLFK TASDDWVLLN VNVTGYFQVN YDEDNWRMIQ HQLQTNLSVI 651 PVINRAQVIY DSFNLATAHM VPVTLALDNT LFLNGEKEYM PWQAALSSLS 701 YFSLMFDRSE VYGPMKKYLR KQVEPLFQHF ETLTKNWTER PENLMDQYSE 751 INAISTACSN GLPQCENLAK TLFDQWMSDP ENNPIHPNLR STIYCNAIAQ 801 GGQDQWDFAW GQLQQAQLVN EADKLRSALA CSNEVWLLNR YLDYTLNPDL 851 IRKQDATSTI NSIASNVIGQ PLAWDFVQSN WKKLFQDYGG GSFSFSNLIQ 901 GVTRRFSSEF ELQQLEQFKK NNMDVGFGSG TRALEQALEK TKANIKWVKE 951 NKEVVLNWFI EHS
Affidavit
I herewith declare that I autonomously carried out the dissertation entitled “Analysis of viral and host factors influencing Alphacoronavirus life cycle in chiropteran and porcine cell lines”.
No third party assistance has been used.
I did not receive any assistance in return for payment by consulting agencies or any other person. No one received any kind of payment for direct or indirect assistance in correlation to the content of the submitted thesis.
I conducted the project at the following institute: Institute of Virology, University of Veterinary Medicine Hannover.
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a similar context.
I hereby affirm the above statements to be complete and true to the best of my knowledge.
____________________________
Acknowledgement
Firstly, I would like to express my deepest gratitude to my supervisor PD, Dr.
Christel Schwegmann-Weßels, for providing me the opportunity to work on this fascinating topic as well as for her continuous guidance, support, patience, motivation, and knowledge.
I would also like to thank Prof. Dr. Andreas Beinecke and PD, Dr. Eike Steinmann,
I would also like to thank Prof. Dr. Andreas Beinecke and PD, Dr. Eike Steinmann,