Design of JEV chimeric glycoprotein constructs using a VSV-G template

In an attempt to induce an interaction between the JEV envelope glycoproteins and retroviral

gag-pol cores in pseudotype production, the construction of several chimeric glycoproteins

was attempted. These maintain the E ectodomain of JEV on the external, extraviral surface, but the transmembrane domains (TMDs) of JEV are altered by introduction of, or swapping with, the TMD and cytoplasmic tail (C-tail) of vesicular stomatitis virus G protein (VSV-G). A VSV-G template was chosen to be employed in the production of JEV chimeric glycoproteins, since this envelope glycoprotein pseudotypes retroviral cores with high efficiency and has been shown to enhance the capability of other virus envelopes producing functional HIV pseudotypes (Carpentier et al, 2012). The following JEV prME/VSV-G chimeric glycoproteins (cGP) constructs were considered for design and utilisation in JEV pseudotyping experiments (see Figure 22 for schematics of all constructs):

Chimeric glycoprotein #1: Replacing second JEV E TMD with the C-tail of VSV-G

The simplest chimeric glycoprotein approach involves the substitution of the second JEV E TMD with the C-tail of VSV-G. The VSV-G C-tail is essential for its interaction with retroviral gag

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and subsequent glycoprotein coating during pseudotype production, so has been included in this cGP to stimulate similar connections for JEV envelope glycoproteins. However, a main concern with this cGP construct is that pseudotype viruses have to successfully engage with target cells and induce entry via membrane fusion for VSV-G, it has been confirmed that its TMD is vital for fusion with permissible target cell membranes (Cleverley and Lenard, 1998), whereas the unique hairpin motif between the two flavivirus TMDs is crucial for fusion and cellular entry for this family of viruses (Fritz et al, 2011). The absence of the complete TMD segment from either virus may prevent the success of this cGP construct.

Chimeric glycoprotein #2: Replacing both of the JEV E TMDs with the TMD and C-tail of VSV-G

JEV/VSV cGP #2 involves the replacement of both JEV TMDs with the VSV TMD and C-tail. Although this construct possesses the intact VSV transmembrane and cytoplasmic sections so should be fusion competent, the unique feature of flavivirus envelope proteins is that they contain a double TMD with a hairpin. It is possible that interactions between the ectodomain and transmembrane regions of JEV could be hindered by the removal of the distinctive JEV TMD segment. Furthermore, heterodimeric interactions between JEV prM and E proteins have been previously reported (Lin and Wu, 2005; Peng and Wu, 2014), and the absence of the E TMDs from this chimeric glycoprotein may prevent their intra-membrane contact with the prM transmembrane residues, possibly resulting in reduced prM-E heterodimerisation and

inhibition of JEV structural conformational change and maturation.

Chimeric glycoprotein #3: Appending the VSV-G TMD and C-tail onto the C-terminus of full length, wild-type JEV E

The third cGP involves appending the VSV-G TMD and C-tail onto the C-terminus of the full- length JEV E protein. This construct will result in the expression of three TMDs: the JEV E double TMD hairpin followed by the VSV-G TMD. The presence of the intact JEV

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transmembrane segment should allow for interactions between E and prM, and subsequent virion envelope maturation; interaction with HIV or MLV gag proteins, and in turn pseudotype virus formation should occur, owing to the VSV-G TMD/C-tail. If successful PV production indeed takes place, the pseudoparticles should be fusion-competent with target cell

① ① ① ① ① ① ①H ① ① ①

disadvantage of this construct is that the addition of an extra, heterologous transmembrane- spanning domain may physically block and hinder intra-membrane prM-E interactions.

Chimeric glycoprotein #4: Appending a duplicate first JEV E TMD and the VSV-G C-tail onto the C-terminus of wild-type, full length JEV E

Chimeric glycoprotein construct #4 is similar to #3, but involves the duplication of the first JEV E TMD, in place of the VSV-G TMD, whilst retaining the VSV-G C-tail. This approach may create lower levels of interaction inhibition between the prM and E TMDs, as the extra domain embedded in the membrane is native to JEV. Nonetheless, the presence of this duplicated TMD may cause steric hindrance and subsequent envelope protein conformational distortion, as well as potentially inducing incorrect interactions with other prM and E TMDs.

Chimeric glycoprotein #5: Inserting an anti-parallel tandem repeat of the VSV-G C-tail between the two JEV E TMDs

JEV/VSV cGP #5 involves the insertion of an anti-parallel tandem repeat of the VSV-G cytoplasmic tail between the two JEV E TMDs. As the VSV-G C-tail is all that is required to interact with retroviral core proteins during pseudotype assembly, it is possible that the TMD is obsolete for chimeric glycoprotein construction, as fusion of pseudoparticles into target cells can be mediated by the full JEV E TMD segment. If this is the case, then construct #5 would not alter the transmembrane topology of the JEV envelope proteins. As the VSV-G C-tail is

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formation and attachment of its C-terminus to the second JEV TMD, restricting its interaction with retroviral gag. Inclusion of two anti-parallel C-tails should enable them to sit adjacent to one another and more exposed to the cytosol, facilitating interaction with the pseudotyping cores.

1 6 0 VSV-G JEV E 1 2 #1 1 #2 #3 1 2 #4 1 2 1 #5 1 2

Figure 22. A selection of schematics to represent the five JEV/VSV chimeric glycoprotein constructs. #1) Replacing second JEV E TMD with the C-

tail of VSV-G. #2) Replacing both of the JEV E TMDs with the TMD and C-tail of VSV-G. #3) Appending the VSV-G TMD and C-tail onto the C- terminus of wild-type, full length JEV E. #4) Appending a duplicate first JEV E TMD and the VSV-G C-tail onto the C-terminus of wild-type, full length JEV E. #5) Inserting an anti-parallel tandem repeat of the VSV-G C-tail between the two JEV E TMDs. Cartoon depictions of the native VSV-G

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