Identification of viral protease-dependent cleavage sites within the human astrovirus polyprotein

Infection by human astrovirus (HAstV), a small, positive-strand RNA virus, is a major cause of gastroenteritis and has been implicated in an increasing number of severe, sometimes fatal, neurological diseases since 2008. Currently, there are no vaccines or antiviral treatments available to treat HAstV infection. An attractive target for antiviral therapeutics is the viral protease due to its essential functions throughout infection. However, the molecular mechanisms of the HAstV protease, nonstructural protein 1a/3 (nsp1a/3), are poorly understood. In fact, the specific residues within the cleavage junctions that are targeted by nsp1a/3 during polyprotein processing have yet to be experimentally identified. To identify the junctions between viral proteins, we performed mass spectrometry and site-directed mutagenesis using epitope-tagged viral polyprotein expression plasmids. Using these strategies, we identified a consensus motif that is found throughout the polyprotein near previously proposed junctions. We found that cleavage occurs after a hydrophobic residue - X - Gln motif. Further mutagenesis of surrounding sequence identified the importance of basic residues following the motif for efficient processing. Cleavage at each junction was determined to be essential for the production of progeny virions. However, abolishing nsp1a/4-VPg cleavage allowed efficient replication, suggesting that VPg can function in an intermediate form. Overall, our results identify a conserved cleavage motif that is recognized by the nsp1a/3 protease within the viral polyprotein, and cleavage at this motif was found to be essential for the recovery of progeny virions. These findings will be instrumental in further understanding the basic functions of HAstV polyprotein processing during infection.IMPORTANCEHuman astroviruses (HAstVs) are a leading cause of non-bacterial gastroenteritis in children, elderly individuals, and immunocompromised patients. However, infection by divergent strains of HAstV is now recognized as a causative agent of severe neurological diseases, which can have fatal outcomes. Despite the global prevalence of HAstV, we currently have a limited understanding of the biology of these viruses. Translation of the viral genome leads to the production of polyproteins that are processed by viral and host proteases into functional proteins. In this study, we identified a conserved recognition sequence targeted by the viral protease for cleavage. Importantly, these findings elucidate the N- and C-termini of the nonstructural proteins within the HAstV polyprotein, offering valuable information for future studies on the function of individual viral proteins. Similar to other positive-sense RNA viruses, the necessity of proteolytic processing for the HAstV polyprotein highlights the viral protease as a promising target for antiviral development.