The porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus stands as one of the most impactful infectious pathogens, causing substantial economic losses in the swine industry. The minor glycoprotein Gp3 possesses an unusual hairpin-like membrane topology. The N- and C-terminus are exposed to the outside of the virus particle and anchoring to the membrane is achieved by an amphiphilic helix. The objectives of this study were to investigate the influence of the amphiphilic helix and the C-terminus of GP3 on viral replication in cell culture and to investigate whether the amphiphilic helix can be replaced by another amphiphilic helix with the same properties. I demonstrate that deletion of the C-terminal region leads to a slowdown in virus growth compared to the wild-type virus. Notably, the virus regains the C-terminus of GP3 after serial passage in cell culture. Moreover, substituting amino acids in the hydrophilic face of the helix with alanine results in the failure to rescue the virus, while the exchange of hydrophilic amino acids with more hydrophobic ones either causes virus rescue failure or strong reduction in virus titer. Additionally, swapping the position of two pairs of amino acid within the amphiphilic helix, which maintains its hydrophobicity and amphiphilic character, prevents rescue of infectious virus particles. Intriguingly, despite these alterations, the amphiphilic helix with substituted amino acids retains its binding capacity to membranes. This underscores that the amphiphilic helix cannot be replaced by another amphiphilic helix with similar biophysical properties without affecting the virus's behavior. Finally, five inhibitors were tested on PRRSV-1 and PRRSV-2 replication and it was shown that Remdesivir, GS-441524, EIDD-2801 and Ribavirin effectively inhibit virus replication whereas GC376 have no effect.
