Location and putative function 114

The two point mutations, G52R and T173I, are located in two separate positions in the YFV E protein. Although the crystal structure of YFV E protein has not been described, based on the homology of the amino acid sequences among flaviviruses, the G52R mutation is considered to be part of the molecular hinge structures between EDI and EDII. (Table 4.2) There are four molecular hinge peptides (designated as H1-H4) which exist as loosely packed structures in the junctions between EDI and EDII of flavivirus E proteins (Rey, Heinz et al. 1995, Modis, Ogata et al. 2004). The sequences of the four amino acids in the H1 region which harbors the G52R mutation of YFV 17D strain show a high degree of variation in the first and second amino acid residues among flaviviruses. Regardless of the virus serocomplex and vector type, the last two amino acids of the hinge region, are characterized by having high hydrophobic sidechains. The third amino acid residue is predominantly composed of proline and leucine, although DENV-4 utilizes valine. The fourth amino acid residue typically is the small hydrophobic alanine except for the use of proline or threonine in three flaviviruses (ENTV, SEPV, YOKV) that have not been isolated from arthropod vectors but are classified under the YFV serocomplex based on the genetic homology.

Serocomplex Virus strain Sequence of H1 peptide (E 51-54)

YFV YFV Asibi DGPA

YFV 17D DRPA

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SEPV MK7148 DTPT

YOKV Oita36 NNPP

DENV DENV-1 Hawaii TNPA

DENV-2 New Guinea C KQPA

DENV-3 H87 TQLA DENV-4 H241 KEVA JEV JEV SA14 SQLA JEV SA14-14-2 SQLA WNV NY99 ANLA KNV MRM61C ANLA SLEV MS1-7 TELA MVEV MVE1-151 TNLA

TBEV TBEV Neudoerfl ENPA

OHFV Bogluvovska ENPA

Table 4.2 Sequences of the molecular hinge H1 peptide between flavivirus EDI and EDII The hydrophobic amino acids are labeled by underline.

Although the four molecular hinge peptides are often displayed as four distinct regions in the alignment of full-length E proteins, due to the discontinuous nature of the numbering of the residues of EDI and EDII, the peptides are consistently located in close proximity to the three- dimensional structure of flavivirus E proteins. The location and overall structure of the molecular hinge region is displayed in the crystal structure of the DENV-2 E protein dimer as shown in Figure 4.1. The G52R mutation exists in the H1 molecular hinge between the β-strand D0 of EDI

and β-strand a of EDII. The region has been hypothesized to accommodate the conformational change between EDI and EDII during viral membrane fusion. The detail mechanisms of viral membrane fusion and the crystal structure and functional characterization of four peptides in the molecular hinge will be discussed in section 4.1.B and 4.1.C.

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Figure 4.1 The location of the molecular hinge region in the crystal structure of DENV-2 E protein dimer (PDB ID: 1OAN)

The individual domains of DENV-2 E protein are coded in red for EDI, yellow for EDII and blue for EDIII. The four short peptides consist of the molecular hinge region are labeled in cyan.

The G52R mutation is a conserved between the 17D-204 and 17DD substrains used for vaccine production. The H1 molecular hinge of the FNV strain showed conservatation of the mutation at the forth amino acid residue compared to the parental FVV. The sequence alignment of the YFV E proteins is summarized in table 4.3.

Strain Sequence of H1 molecular hinge

Asibi DGPA

17D-204 DRPA

17DD DRPA

FVV DGPA

FNV DGPV

Table 4.3 Sequences of H1 molecular hinge region in YFV E proteins of the wildtype virulent strains and the attenuated vaccine strains

The T173I mutation is located in the G0H0 loop structure between the G0 and H0 β-

strands of YFV EDI. The G0H0 loop structure is composed of the conserved “glycine-tyrosine-

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formation of G0 and H0 β-strands observed in all flavivirus E proteins. The relative location of

the G0H0 loop structure of flavivirus EDI is displayed in the crystal structure of DENV-2 E

protein dimer in Figure 4.2.

Figure 4.2 The relative location of the G0H0 loop in the crystal structure of DENV-2 E protein dimer (PDB ID: 1OAN)

The three domains are labeled in red for EDI, yellow for EDII and blue for EDIII. The G0H0 loop structure is labeled in cyan.

The flavivirus EDI serves as a structurally central domain which connects EDII and EDIII. EDI contains two β-sheets that contributes to the formation of the overall β-barrel structure. The G0H0 loop is located between the neighboring antiparallel G0 and H0 β-strands as

displayed in Figure4.3. With the additional B0 and I0 β-strands, the inner B0I0G0H0 β-sheet faces

the viral membrane underneath the outer A0C0D0E0F0 β-sheet. The two β-sheet structures are

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Figure 4.3 The relative location of the G0H0 loop structure in the crystal structure of DENV-2 E protein monomer (PDB ID: 1TG8)

EDI is labeled in red with two neighboring domains, EDII in yellow and EDIII in blue. The β-barrel structure of DENV-2 EDI consists of two β-sheets structures. The B0I0G0H0 β- sheet faces the viral membrane and contains the G0H0 loop labeled in cyan. Five β-strands form the A0C0D0E0F0 β-sheet forms as the external surface.

The structurally central domain, EDI does not undergo extensive structure rearrangement during the fusion process. In addition to the presence of several B-cell epitopes that have been reported on the G0H0 loop in EDI of wildtype YFV and DENV, the YFV EDI is different from

the DENV EDI due to the absence of the Asn67 glycosylation site that has been characterized as the binding site of DC-SIGN (Gould, Buckley et al. 1989, Pokidysheva, Zhang et al. 2006, Lai, Goncalvez et al. 2007, Fibriansah, Tan et al. 2014). The details of the B-cell epitopes in the G0H0

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suggested that YFV uses amino acid residues that are largely conserved among other flaviviruses as summarized in table 4.4.

Serocomplex Virus Strain Sequence of G0H0 loop

YFV YFV Asibi EAEFTGYGK

YFV 17D EVEFIGYGK

ENTV UgIL-30 VVVFTGYGT

SEPV MK7148 TVTFTGYGN

YOKV Oita36 VIGFAGYGT

DENV DENV-1 Hawaii EIQLTDYGA

DENV-2 New Guinea C EAELTGYGT

DENV-3 H87 EAILPEYGT

DENV-4 H241 EVKLPDYGE

JEV JEV SA14 TLKLGDYGE

JEV SA14-14-2 ALKLGDYGE

WNV NY99 TLKLGEYGE

KNV MRM61C TLKLGEYGE

SLEV MS1-7 TANMGEYGT

MVEV MVE1-151 TAKMGDYGE

TBEV TBEV Neudoerfl ILTMGEYGD

OHFV Bogluvovska ILTMGEYGD

Table 4.4 Sequences of the G0H0 loops in EDI of flaviviruses

The amino acid residues in the G0H0 loops are underlined based on the available three- dimensional structures and the sequence homologies.

The consistent use of glycine or proline as the amino acid residues in the flavivirus G0H0

loop structure follows the biochemical properties of the secondary turn structures (Lodish 2013). However, minor structural difference remains in flaviviruses in YFV serocomplex, DENV-1 and DENV-2. The G0H0 loops of YFV-serocomplex flaviviruses, DENV-1, DENV-2 and DENV-3

are consistently shorter than the loops of members of the JEV and TBEV serocomplexes. The elongated structure of JEV- and TBEV-serocomplex flaviviruses is due to the insertion of an aspartate or glutamate residue following the first turn made by the glycine residue, and an

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aspartate or glutamate residue prior to the H0 β-strand. The comparison was made utilizing the

available crystal structures of DENV-2, DENV-3, JEV and TBEV E proteins. (Fig. 4.4)

Figure 4.4 The G0H0 loop structure in flavivirus EDI

EDI of four different flaviviruses are displayed in (a) DENV-2 (PDB ID: 1TG8), (b) DENV- 3 (PDB ID: 1UZG), (c) JEV (PDB ID: 3P54), and (d) TBEV (PDB ID: 1SVB). The G0H0 loop structure is labeled in cyan with residues from EDI in red, EDII in yellow and EDIII in blue.

The T173I mutation is a conserved mutation between the 17D-204 and 17DD vaccine substrains. As the wildtype YFV-specific B-cell epitope, the threonine residue is conserved among natural isolates of YFV. The plaque purification of the 17D-204 substrain demonstrated maintenance of a minor viral population that use a threonine residue in the G0H0 loop structure.

This was recognized by the wildtype-specific mAb 117 (Gould, Buckley et al. 1989). The details regarding to the phenotype of the mAb 117-reactive variants in vivo will be discussed in section 4.1.D.

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