Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus

Abstract

We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin ␣ 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (␣ 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing ␣ 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

“…Furin-adapted a 1 -ATs have been shown to efficiently inhibit the formation of infectious progeny of other viruses (e.g. HIV, measles virus and human cytomegalovirus) [38,39,40,41,68,69]. Using a replication-competent recombinant VSV pseudotyped with the LASV glycoprotein GP [48], we demonstrate that proteolytic maturation of the precursor GP-C is sensitive to S1Padapted a 1 -ATs.…”

“…Due to the introduction of a second mutation from alanine to arginine at P4 of the RCL, the engineered a 1antitrypsin variant Portland (a 1 -AT-PDX) showed high affinity for furin [38]. a 1 -AT-PDX efficiently inhibited the formation of infectious HIV, measles virus, and human cytomegalovirus progeny by blocking furin-dependent processing of glycoproteins gp160, F0 and gB, respectively [38,39,40,41]. Pullikotil and coworkers used this approach for the generation of highly selective a 1 -antitrypsin variants specific for S1P by introducing various S1P recognition motifs into the RCL of a 1 -antitrypsin [42].…”

Biochemical and biophysical comparison of cleaved and uncleaved soluble, trimeric HIV-1 envelope glycoproteins

“…Of particular note is that the crystal structure of the uncleaved form of the human H1 hemagglutinin (HA) from Influenza virus showed that the cleavage event only minimally alters the local structure of the transmembrane glycoprotein and its antigenic sites (Stevens et al, 2004). In addition, in vitro and in vivo studies on several viruses have shown that Env glycoprotein cleavage is not always necessary for virus maturation/assembly or infectivity, implying that cleavage has little effect on function (Bos, Luytjes, and Spaan, 1997;Hingley, Leparc-Goffart, and Weiss, 1998;Russell, Dalrymple, and Johnston, 1989;Watanabe et al, 1995). However, these conclusions are often derived from one-step infection assays; it is possible that additional selection pressures are exerted in multiple replication rounds, and/or in vivo, that result in the retention of the highly conserved cleavage motif.…”

Curbing activation: proprotein convertases in homeostasis and pathology

“…The fusion protein of measles virus is produced by processing of a precursor (F 0 ) at a R-R-H-K-R sequence (46). Expression of correctly processed F-protein in Vero cells leads to membrane fusion and formation of syncytia.…”