Abstract
Human norovirus (HuNV) infections are a major cause of gastroenteritis, with an associated cost of $60 billion worldwide. HuNV can be cultured in vitro, but growth in these systems remains challenging. As a result, little is known about HuNV host-cell interactions or viral proteins. Therefore, murine norovirus (MNV) is often studied as a model system due to the ability to analyse active infections in a routine cell line.
The norovirus non-structural protein 1-2 (NS1/2) is a poorly conserved, disordered, protein that has an important role during viral replication. Evidence has shown that MNV NS1/2 can be processed into sub-fragments by the host apoptotic caspases-3 and 7. HuNV NS1/2 has also been shown to be cleaved by caspase-7 in vitro. However, MNV undergoes pyroptosis as well as apoptosis and it is unknown whether the other host caspases cleave NS1/2. The function of NS1/2 or its caspase-derived sub-fragments is mostly unknown.
This study aimed to first express and purify MNV and HuNV NS1/2 and investigate the ability of the pyroptosis-induced caspase-1 and the third apoptosis executioner caspase-6 to process them in vitro. Phosphorylation’s effect on caspase processing of NS1/2 was also investigated. To confirm processing in a cellular context, NS1/2 cleavage was analysed by either MNV infection, or HuNV NS1/2 expression through baculovirus transduction, where caspases were activated via the induction of either apoptosis or pyroptosis. Finally, a sub-fragment of HuNV NS1/2 will be expressed in a recombinant baculovirus system, to provide a platform for further investigations into the characterisation of NS1/2 fragments.
A recombinant baculovirus system was used to express and purify 50 mg/L and 64 mg/L of HuNV GII.4 Sydney 2012 and MNV-1 NS1/2 or regions thereof respectively. In vitro caspase activity assays confirmed that caspase-3 and 7 cleaved MNV NS1/2, and caspase-7 cleaved HuNV NS1/2. Interestingly, HuNV and MNV NS1/2 are also processed by caspase-1 and caspase-6 in vitro. Cleavage of HuNV NS1/2 by capsases-1, 6 and 7 generated four fragments of similar size (~29 kDa, ~19 kDa, ~16 kDa and ~7 kDa) indicating conserved caspase cleavage points within HuNV NS1/2. A 6.6 kDa HuNV NS1/2 fragment generated by caspase-1 cleavage was identified via mass spectrometry. A recombinant baculovirus system was used to express the 6.6 kDa fragment in insect cells where it was shown to be soluble. The phosphorylation state of either NS1/2 protein had no effect on caspase activity in vitro. MNV NS1/2 as well as ~25 kDa and ~15 kDa sub-fragments, were expressed and detected in infected RAW264.7 cells. The generation of the MNV NS1/2 fragments could be enhanced to some extent by the early induction of apoptosis. HuNV NS1/2 was able to be expressed and detected via transduction of HEK293 cells.
This project implies that NS1/2 is processed by alternative caspases, generated by the induction of apoptosis or pyroptosis into more sub-fragments than previously known. Our evidence suggests there are caspase cleavage sites within NS1/2 conserved across various caspases involved in unique cell death pathways, indicating a possible functional role of fragments produced by this cleavage in the noroviral lifecycle. With the successful expression of the 6.6 kDa fragment, this work also provides a foundation for further characterisation of NS1/2 fragments.