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.
Bibliographical noteFunding Information:
We thank J. Aronson (University of Texas Medical Branch) for providing the stock P2 and P18 viruses, K. Conzelmann (Ludwig-Maximilians-Universit?t, Germany) for the BSRT7-5 cells, J. Nunberg (University of Montana, USA) for the T7 promoter-directed firefly luciferase plasmid, and E. Hunter (Emory University, USA) for anti-p24 antibody.This work, including the efforts of Yuying Liang, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI083409). This work, including the efforts of Hinh Ly, was funded by HHS|NIH| National Institute of Allergy and Infectious Diseases (NIAID) (AI093580).