Characterization of a secreted form of measles virus haemagglutinin expressed from a vaccinia virus recombinant

Abstract

The measles virus (MV) haemagglutinin (HA) is a class 2 glycoprotein by means of which the virus particle attaches to the host cell receptor. We have previously expressed this glycoprotein as a vaccinia recombinant virus and have shown that the HA glycoprotein synthesized is indistinguishable from that coded by MV. In the present study, we report that in RK13 cells a soluble form (sHA) of the HA is secreted into the medium. We show by SDS-PAGE and sucrose density gradient centrifugation that the sHA is a dimer and is smaller than the cell-associated form. Using a variety of inhibitors the production of sHA was shown to be a late event, probably occurring at the membrane; only fully glycosylated molecules were found in sHA. Finally, we demonstrate that sHA retains its antigenicity with conformation-dependent MAbs and its receptor recognition function. We conclude that sHA is a valuable tool for use in studies of the structure and function of the MV HA glycoprotein.

Ectodomain shedding of the glycoprotein GP of Ebola virus

“…Soluble glycoproteins have been described for a number of viruses (Morimoto et al, 1993;Malvoisin and Wild, 1994;Roberts et al, 1994), but little is known about the mechanisms and the biological significance of their release. Inhibitory effect on GP 1,2D shedding TAPI-I (50 mM) + 1,10-Phenanthroline (1 mM) a + EDTA (5 mM) À EGTA (5 mM) À Phosporamidon À b a-1-Antichymotrypsin (4 mM) À Captopril (10 mM) À BB2516 (500 nM) + GM 6001 (5 mM Premature termination of translation has been suggested to be responsible for release of the carboxy-terminally truncated G protein of VSV (type I transmembrane glycoprotein) (Grü nberg et al, 1991).…”

A Subset of Porcine Reproductive and Respiratory Syndrome Virus GP 3 Glycoprotein Is Released into the Culture Medium of Cells as a Non-Virion-Associated and Membrane-Free (Soluble) Form

“…By using monomer-and oligomer-specific antibodies to influenza virus hemagglutinin, it was shown that monomers are restricted to the ER, whereas assembled molecules are able to reach distal points in the transport pathway (9). Furthermore, a fraction of the hemagglutinin of measles virus expressed from a vaccinia virus recombinant was found to be released into the culture medium as a dimer (35). These are but a few examples emphasizing the importance of the oligomeric assembly in the efficient transport of viral glycoproteins, which may be regarded as a likely explanation for the retention of the PRRSV GP 3 in the ER.…”

“…Confluent MARC-145 cells growing in 150-cm 2 flasks (3 ϫ 10 7 cells) were infected with 10 7were infected with AdCMV5/ORF3 at an MOI of 20 to 30 TCID 50 /cell. At 24 h p.i., cells were pulse-labelled with [ 35 ]methionine for 30 min (time zero), chased for the indicated times, and processed for radioimmunoprecipitation with ␣3 antiserum. Aliquots of immunoprecipitates were treated with Endo H or Glyco F or were left untreated (UNT) and were then analyzed by SDS-12% PAGE.…”

Preparation of Recombinant Viral Glycoproteins for Novel and Therapeutic Antibody Discovery

“…A useful approach to develop viral membrane glycoproteins suitable for panning phage libraries or as antigens for eliciting antibody responses that recognize their native form is to engineer soluble and secreted versions of the molecules. Often, this approach yields a quaternary structure similar to their native counterparts, and for animal viruses eukaryotic expression systems are typically employed such as recombinant bacculovirus or vaccinia virus, or transient or stable expression in cell culture (22,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). This chapter will detail several methods that have been successfully employed to produce, purify, and characterize soluble and secreted versions of several viral envelope glycoproteins which have been successfully used as antigens to capture and isolate human phage-displayed mAbs.…”