Abstract
Human cytomegalovirus (HCMV) is a β-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.
Highlights
Human cytomegalovirus (HCMV) is a -herpesvirus that has co-evolved with the host immune system to establish lifelong persistence
We have reported the crystal structure of the HCMV immunomodulatory protein UL144 bound to BTLA
The BTLA contact residues employed by UL144 or HVEM–BTLA complex are shown as yellow (HVEM) differ significantly, supporting a model where UL144 has evolved uniquely to interact with this immune inhibitory molecule
Summary
The design of expression constructs for both BTLA and UL144 is based on the domain architecture shown in Fig. 1, A and B. Because the ectodomain of UL144 contains a total of eight putative N-linked glycosylation sites that were spread across CRD2 and the membrane extension region, expression of UL144 in mammalian HEK293T cells yielded a heavily-glycosylated protein that we assumed would impede subsequent crystallization. The CRD1 and the majority of the CRD2 region of UL144 are ordered, we have not observed obvious electron density corresponding to the membrane extension region (residues 96 –132) in any of the four copies This may reflect a greater flexibility in this region that links the UL144 ectodomain to the membrane. Superposition of all four copies of the UL144 –BTLA complex indicated high-similarity in the structure of BTLA and the CRD1 region of UL144 (root mean square deviation value (RMSD) of 0.124 Å) (Fig. S1A). The structure of the CRD2 region of UL144 exhibited slight differences between the four copies within the asymmetric unit, likely resulting from crystal packing. Superposition of all four individual UL144 molecules yielded a similar overall structure with an RMSD value of less than 1 Å across all C␣ atoms (Fig. S1B)
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