Speaker
Description
Human cytomegalovirus (HCMV) employs multiple viral glycoproteins to regulate cell entry, intracellular trafficking and immune evasion. The immunomodulatory glycoprotein UL141 interacts with the viral envelope glycoprotein gH and the cellular death receptor TRAIL-R2. However, the structural basis of these interactions and their potential modulation by synthetic ligands remain poorly understood. This study aimed to establish a platform for the biochemical and structural characterization of the HCMV UL141–gH complex as a prerequisite for investigating the competitive and allosteric interactions of UL141 with gH, TRAIL-R2 and the synthetic glycomimetic TK219.
Soluble ectodomains of Strep-tagged gH and His-tagged UL141 were co-expressed in suspension-adapted HEK293F cells. The complex was isolated using sequential His- and Strep-affinity chromatography, proteolytic tag removal and size-exclusion chromatography. Co-elution during size-exclusion chromatography and the presence of both proteins in the purified fractions confirmed formation of a stable heterodimeric complex. SDS-PAGE revealed bands corresponding to glycosylated gH and UL141 at approximately 78–90 and 38–48 kDa, respectively. Dissociation into individual subunits under non-reducing SDS-PAGE conditions indicated that the complex is stabilized by non-covalent interactions.
Far-UV circular dichroism spectroscopy demonstrated that the purified complex is folded and exhibits a mixed α/β secondary-structure profile. Negative bands near 208 and 222 nm indicated a substantial α-helical contribution, predominantly attributable to gH, whereas UL141 likely contributed to the broader signal within the 215–220 nm region. Initial sitting-drop crystallization screening produced crystals under several conditions. Synchrotron diffraction tests at the ESRF MASSIF-1 beamline yielded diffraction extending to approximately 2.42 Å. However, incomplete low-resolution reflections and ambiguous indexing prevented reliable structure determination. These results establish a reproducible procedure for producing folded UL141–gH complex and provide a foundation for further crystallographic optimization and quantitative competition studies examining whether TK219 modulates the mutually exclusive interactions of UL141 with gH and TRAIL-R2.
Acknowledgement
Financial supports provided by the Slovak Research and Development Agency (APVV-19-0376 and APVV-24-0351) and the Scientific Grant Agency of the Slovak Republic (VEGA-02/0049/26) are gratefully acknowledged.
