Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation

Abstract

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus–host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro . IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

Structure and antigenicity of the divergent human astrovirus VA1 capsid spike

“…Residues 297–347 that were not identified by mass spectrometry map to this outer region, suggesting that they are accessible on the surface of the assembled virus capsid for protease cleavage. It is interesting to note that residue E296, the last VP33 residue observed by mass spectrometry, is in a structurally similar location as a putative trypsin maturation cleavage site in classical HAstVs (R313 in HAstV-8) that is thought to be important for exposing a membrane-lytic peptide ( Fig 4C and 4D ) [ 59 ]. Next, we observed that VP38 amino acids include the C-terminal end of the predicted HAstV-VA1 capsid core structural domain, a predicted linker region, and the full spike domain ( Fig 4C ).…”

“…Specifically, our mass spectrometry data support that the HAstV-VA1 capsid becomes cleaved in one or more places in the outer region of the capsid core domain, which forms the shell of the virus capsid. Recent studies suggest that this proteolytic processing in HAstVs may expose a membrane-lytic peptide utilized for host membrane disruption during cell entry [ 59 ]. Our data support that HAstV-VA1 utilizes a similar mechanism to promote infectivity.…”