Human parainfluenza virus type 3 viruses with the furin-susceptible motif at the cleavage site of the fusion protein arose from original wild strains during their propagation in vitro

Human parainfluenza virus type 3 (HPIV3) requires proteolytic cleavage of its fusion proteins by either exogenous serine proteases or a ubiquitous intracellular protease, furin, for replication in vitro. Viruses that utilize furin possess furin-susceptible amino acid sequence motifs at the cleavage site of the fusion protein when they infect host cells; without the motif, the virus cannot replicate in cultured cells without the aid of serine proteases such as trypsin. While HPIV3 isolates carrying this furin-susceptible motif (VWFM) were frequently reported in studies conducted before 1990, they are now largely regarded as laboratory-adapted variants and are not detected in viruses within clinical specimens. Although this artifactual nature has been widely postulated, definitive experimental evidence detailing the mutation process has been limited. Building on this concept, we hypothesized that the VWFM is artificially selected during the culturing of wild viral isolates in the host cell under low-trypsin concentration conditions. We repeatedly passaged HPIV3 strains lacking the furin motif under conditions of low or no trypsin supplementation in cell culture. During this process, we observed the emergence of VWFM, which arose from a single nucleotide substitution at the cleavage site of the F gene and acquired the ability to replicate without exogenous trypsin. Thus, we experimentally demonstrated the occurrence of an amino acid substitution at the cleavage site under selective pressure in vitro. These findings substantiate previous insights, confirming that the VWFM is unlikely to dominate in nature but rather arises artificially during the propagation of HPIV3 in cell culture. (248/250 words).