The norovirus non-structural protein NS1 is generated via caspase-mediated cleavage of NS1-2 during norovirus infection. NS1 is a poorly conserved, intrinsically disordered viral protein. Previous work has shown NS1 to be secreted via unconventional mechanisms in the persistent murine norovirus (MNV) strain CR6 upon infection and has been proposed to modulate the innate immune response. The mechanism underlying NS1 secretion is unknown, as is whether NS1 from an acute strain of MNV (CW1) or from Human norovirus (HuNV) is generated or secreted.
To investigate NS1 secretion, we employed immunoprecipitation techniques to detect secreted MNV NS1 during infection. Additionally, an infection-free cell-based assay utilizing a split luciferase enzyme (HiBit) was developed to identify recombinantly expressed extracellular NS1 proteins from MNV and HuNV. Biochemical inhibition of various unconventional secretion pathways was also performed to elucidate the mechanism of NS1 release.
Our findings reveal that NS1 from both MNV CW1 and HuNV GII.4 is secreted unconventionally independent of infection. Notably, the secretion of NS1 is significantly increased under serum starvation conditions. Furthermore, inhibition of cellular GTPases and autophagy pathways resulted in a marked decrease in the levels of secreted NS1.
This study demonstrates that NS1 is an unconventionally secreted viral protein with this secretion conserved in acute MNV and in HuNV. The increased secretion under serum starvation and the impact of inhibiting specific cellular pathways highlights potential mechanisms for NS1 release and offer insights into the broader mechanisms of unconventional protein secretion in viral infections.