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

Abstract

Arenaviruses can cause lethal hemorrhagic fevers in humans with few preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within the SSP on viral replication in cell culture and in a guinea pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and therefore could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuated in vitro and in vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but it did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growth in vitro but significantly reduced viral virulence in vivo. Taken together, our data suggest that the GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulence in vivo. IMPORTANCESeveral arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, and no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus GPC that consists of an SSP, the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as in other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a good target for the development of antivirals against deadly human arenavirus pathogens.A renaviruses can cause neurologic or hemorrhagic fever diseases in humans, and few preventive or therapeutic measures are available (1, 2). The most significant arenavirus pathogen is Lassa fever virus (LASV), which causes infections that are endemic to many countries in West Africa, with an estimated 500,000 cases resulting in ϳ5,000 deaths annually (3). Except for Junin virus (JUNV), which has a vaccine, Candid#1, used only in Argentina, no licensed vaccine for human use is currently available for any other arenaviruses. Therapeutic options are limited and depend mainly on supportive care. Ribavirin, a nonspecific antiviral compound, has shown some efficacy ...

Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drug Library

“…To further elucidate the underlying mechanisms associated with prevention of LASV entry via lacidipine, we characterized the viral target of lacidipine by serially passaging LASVrv in the presence of lacidipine. The adaptive mutant was found to be located in the interface of SSP and GP2, which was proven to be the target for a range of fusion inhibitors identified in previous drug screens ( 8 , 12 , 16 , 28 , 32 , 33 , 38 , 41 , 42 , 44 – 46 ). In particular, we recovered an interesting resistance mutation, T40K, in the ectodomain of SSP.…”

Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production

“…The methods for generating WT and NP RNase-deficient PICVs were described previously (37). Viral growth curve analysis was performed as described previously (52).…”

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

“…Binding efficacy was evaluated in the absence and presence of the drugs, and no significant decrease in the number of LASVpv particles bound to the cell surface at 4°C was observed in either drug-treated group (Fig. 3B), suggesting that neither drug interferes with the receptor binding subunit GP1 (38,39).…”