Abstract
Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-β-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gBC777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.
Highlights
Number of inflammation-related diseases, and a major cause of neuro-developmental disorders as well as of immunosenescence [2,3,4]
GB Associates with Rafts at the Plasma Membrane—To investigate whether the lipid domain surrounding glycoprotein B (gB) was derived from membrane microdomains, and to study gB targeting to cholesterol-rich microdomains, a virus-like particle (VLP)-based assay was set up in insect cells
The comparison with the control from Pr55Gag-negative infected cells showed that contamination by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) virions or structured cell debris was undetectable from samples collected at Ն90% cell viability
Summary
Primary Antibodies—Antibodies used were: anti-p24 (mAb HIV anti-p24 clone 39/5.4A, Abcam); anti-gB (mAb clone 2F12, Abcam); anti-biotin (mAb clone DVP., Creative Diagnostics); rabbit polyclonal anti-flotillin 1 (Abcam, catalog number ab41927); anti-transferrin receptor (mAb clone H68.4, Thermo Scientific); and anti-␣1 Naϩ/Kϩ ATPase antibody (mAb clone 464.6, Abcam). Cells were incubated for 18 h in culture medium containing 50 M 2Br-palmitate (Sigma-Aldrich). 108 ARPE cells were mRNA-transfected in culture medium containing 10% dialyzed FBS to express either gB or gBC777A. Analysis of gB Surface Expression—ARPE cells that were mock, gB, or gBC777A mRNA-transfected as above were processed 18 h after transfection for CELISA (procedure described in Ref. 11), and steady-state surface expression was measured with 2F12 mAb. gB surface dynamic trafficking was measured 16 h after mRNA transfection by incubating ARPE cells in 50 mM NH4Cland 2F12 mAb-containing medium for 30 min. Plasma membrane protein fractionation from gB or gBC777A transfectant ARPE cells was performed according to Schindler and Nothwang [27] and used the plasma membrane protein extraction kit (Abcam), following the manufacturer’s instructions. ARPE cells were mRNA-transfected as for the donor cells above, stained supravitally with DAPI and imaged under an inverted microscope (Axio Observer, equipped with an AxioCam MRm CCD camera, Zeiss)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
