Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

Abstract

The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex.

Characterization of the Glycoprotein Stable Signal Peptide in Mediating Pichinde Virus Replication and Virulence

“…In contrast, N20A had a significantly lower level of fusogenic activity, whereas G2A, K33A, F49A, and C57A did not exhibit any obvious fusogenic activity in this assay. Our data are consistent with previous studies, which have demonstrated that K33A, F49A, and C57A lead to a nearly complete loss of fusogenic activity of JUNV GPC (22,24,25). K33 has been shown to play a critical role in mediating membrane fusion (8,22,23).…”

“…1B) and not because of any block(s) in the intracellular trafficking. The differential effects of F49A and C57A on GPC trafficking between our results of PICV GPC and other published studies on JUNV and lymphocytic choriomeningitis (LCMV) GPC (24,29) may reflect the potential topological and/or biological differences between the GPCs. In summary, our data suggest that most (six out of eight) of the SSP conserved residues do not affect the intracellular transport or surface expression of the glycoprotein complex.…”

“…The K33 residue has been shown to play an essential role in GPC fusion by interacting with GP2 to prime the complex for pHinduced membrane fusion (8,22,23). The F49 residue is located within the recently identified FILL sorting signal motif that is required for glycoprotein processing and surface expression (8,24). The C57 residue of GP2, along with its H459, C467, and C469 residues, form a zinc-binding center, which provides the structural basis for association with SSP in the glycoprotein complex in order to modulate optimal membrane fusion activity (25,26).…”

“…Many of these residues have been characterized previously in the context of GPC expression and pseudotyped viruses (8,11,(22)(23)(24)(25)(26)(27)(28); however, their biological roles have not been investigated. Here, we show that multiple SSP conserved residues are essential or critical for viral infection of cell culture and/or guinea pigs by participating in the membrane fusion reaction, and that two residues, N37 and R55, are required for viral virulence by a yet-to-be-determined mechanism.…”

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

“…Several SPs of viral proteins have postcleavage functions. For instance, SP GP-C of lymphocytic choriomeningitis virus and Junín virus (Arenaviruses) precursor glycoproteins C (GP-C) is an essential structural component of mature virions and is required in glycoprotein maturation, cell fusion, and virus infectivity (33)(34)(35). , and 48 h p.i., as indicated.…”