Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity

Abstract

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

“…Unlike influenza viruses, arenaviruses do not have proton channels in their lipid envelope that actively acidify the virion upon endocytosis. However, it has recently been shown for LASV that protons passively cross the viral membrane, which leads to acidification of the virus interior prior to membrane fusion. , Similar observations have been reported for Ebola virus, where acidification of virions leads to viral matrix reorganization . Besides the pH-dependent NP–Z interaction, we observed that Z starts to oligomerize between pH 5 and 5.5.…”

“…A potentially important factor could be the pH, as LASV is taken up via the endosomal pathway. It was recently shown that acidification of Lassa virions occurs before membrane fusion via reorganization of the glycoprotein complex. , A low pH could lead to the dissociation of RNPs from the Z protein lining the inner side of the viral membrane. Therefore, we evaluated the pH-dependence of the NP–Z interaction by incubating the proteins at varying pH values between 5 and 7.5 and subsequently measured NP–Z complex formation by nMS (Figure A).…”

Cellular endosomal potassium ion flux regulates arenavirus uncoating during virus entry

“…Based on these results ( Fig. 7 to 9 ) and those of others ( 26 , 35 , 36 ), we suggest K + influences interactions between internal Z matrix and RNPs ( Fig. 10 ).…”

“…As the GPC-CD164 interaction at low pH is known to promote membrane fusion ( 19 ), we reasoned K + may exert its influence on uncoating by one of three options: (i) by modulating the interaction between external virion component GP1 and its CD164 ligand; (ii) by modulating GPC-CD164-mediated membrane fusion; or (iii) by influencing separation between internal virion components such as RNP and Z (i.e., uncoating). This latter possibility is particularly intriguing, considering a similar mechanism proposed for influenza virus ( 26 ) and the recent demonstration that the arenavirus interior is accessible to ion influx via formation of a glycoprotein-associated envelope pore in response to low pH ( 35 , 36 ).…”

“…Interestingly, recent work suggests that the arenavirus envelope can be breached by H + ions, resulting in acidification of the virion interior, measured using pH-sensitive indicator dyes in the context of lentiviral-based pseudovirus particles ( 35 ). Further to this, it has been proposed that arenavirus envelope permeability is mediated by structural rearrangements of the arenavirus glycoproteins themselves, following interactions with cellular factors at low pH ( 36 ). These structural rearrangements are proposed to lead to the formation of a transmembrane pore, allowing the passage of H + into the virion interior as a critical entry trigger promoting virion uncoating.…”