Characterization and in vitro assembly of tick‐borne encephalitis virus C protein

Abstract

Tick‐borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical–chemical approaches, such as size‐exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid‐like structure with that of dengue C protein.

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles

“…DENVC shows a high tendency to form amorphous aggregates, which has been reported for the attempts to in vitro assembly of NCLPs as well as for isolation of NCs from viral particles purified from cells infected by flaviviruses [ 38 – 40 ]. This occurs probably due to the exposure of hydrophobic surfaces in capsid proteins after interaction with nucleic acids [ 19 , 39 ].…”

“…However, the process of oligomerization and regulation of RNA incorporation during flaviviruses’ nucleocapsid assembly has not yet been described. Although in vitro assembly of the recombinant capsid proteins has been well established for alphaviruses [ 34 – 36 ] and hepatitis C virus (HCV) [ 37 ], similar systems generated contradictory results for flaviviruses such as DENV [ 21 , 25 , 38 ] and TBEV [ 39 , 40 ]. Here we described for the first time a successful methodology for systematically follow recombinant DENVC assembly into NCLPs in vitro .…”

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein

“…The mature form of TBEV C protein consists of 96 residues and it has a similar fold as the C proteins of other flaviviruses (S1 Fig) [30][31][32][33][34]. The TBEV C protein is mainly a dimer both in solution, and within the virion, although higher-order oligomerisation has been suggested to occur [34][35][36][37]. The C protein contains a flexible N-terminus (17 aa) and four α-helices (α 1-α 4 ), with dimerization occurring via antiparallel interactions of the α 2 and α 4 helices [7,[30][31][32][33][34]37,38].…”

Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

“…Homology modelling predicts a similar fold to that of other flavivirus C proteins species (Figure S1) (Ma et al , 2004; Dokland et al , 2004; Shang et al , 2018; Pulkkinen et al , 2018; Poonsiri et al , 2019; Barrass et al , 2021). The TBEV C protein is a dimer both in solution, and within the virion, although higher-order oligomerisation has been suggested to occur in other flaviviruses such as Zika virus (Kiermayr et al , 2004; Kaufman et al , 2020; Tan et al , 2020; Barrass et al , 2021). The C protein contains a positively-charged, flexible N-terminus (17 aa) and four α-helices (α 1 –α 4 ), with dimerization occurring via antiparallel interactions of the α 2 and α 4 helices (Ma et al , 2004; Dokland et al , 2004; Shang et al , 2018; Boon et al , 2018; Pulkkinen et al , 2018; Poonsiri et al , 2019; Barrass et al , 2021).…”

“…The C protein contains a positively-charged, flexible N-terminus (17 aa) and four α-helices (α 1 –α 4 ), with dimerization occurring via antiparallel interactions of the α 2 and α 4 helices (Ma et al , 2004; Dokland et al , 2004; Shang et al , 2018; Boon et al , 2018; Pulkkinen et al , 2018; Poonsiri et al , 2019; Barrass et al , 2021). The α 4 helix contains multiple positively-charged residues, thought to form a charged surface responsible for nucleic acid binding (Samuel et al , 2016; Shang et al , 2018; Kaufman et al , 2020). The other side of the dimer (the α 2 helix or the N-terminus) may be important for nucleic acid or membrane binding, although this facet of C protein function remains under-studied (Markoff et al , 1997; Kofler et al , 2002; Nemésio et al , 2013; Shang et al , 2018).…”