Abstract
The 2019 novel coronavirus (COVID-19) pandemic has resulted in millions of deaths
and severely impacted economies globally (1). The causative virus, severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the Coronaviridae
family and shares 79% sequence identity with SARS-CoV (2). The emergence of
highly mutated strains such as Delta and Omicron has decreased the efficacy of the
rapidly developed vaccines (3), highlighting that a combination of prophylactic
vaccine and a repertoire of direct-acting antivirals are necessitated in the future.
The highly conserved interaction of the SARS-CoV-2 non-structural protein 7 (nsp7)
and 8 (nsp8) serves as a novel antiviral target. These proteins are hypothesised to
function as processivity factors for nsp12 the RNA-dependant RNA-polymerase
(RdRp) within the core polymerase complex (4). Alternatively, research on SARS-CoV
and feline coronavirus (FCoV) led to the hypothesis that nsp7 and nsp8 form a
putative primase complex, responsible for adding nucleotides to a single-stranded
template, forming a primer to which nsp12 (RdRp) can bind to and transcribe the
genome (5-7). Antivirals targeting the conserved alpha-helical regions that govern
the nsp7/nsp8 interaction can potentially disrupt their role as the putative primase
or as processivity factors within the core polymerase complex. Such an antiviral may
be less prone to developing mutation-acquired resistance and have activity against
other coronaviruses.
This thesis details the expression and purification of SARS-CoV-2 nsp7 and nsp8 in a
recombinant Escherichia coli (E. coli) expression system and techniques such as
chemical cross-linking, native PAGE electrophoresis, size exclusion chromatography
and multi-angle light scattering were used to analyse their monomeric and
multimeric nature. Both nsp7 and nsp8 can form dimers and potentially higher
order structures. The formation of an nsp8 dimer likely has increased stability
compared to the monomeric form. We also provide evidence supporting the nsp7-
nsp8 interaction in forming a heterodimer and heterotetramer. Interestingly the
degradation or truncation of nsp8 appears linked to the formation of the nsp7/nsp8
heterotetramer.
This thesis contains three potential avenues for high-throughput screening of
antivirals targeting the nsp7/nsp8 interaction. A fluorescent-based assay to measure
de-novo synthesis of the putative primase complex, which ultimately did not detect
primase activity. An AlphaLISA measuring protein-protein interaction in solution
was hindered by the requirement of protein affinity tags. Finally, an ELISA
measuring the interaction of protein without epitope tags proved capable of
detecting this interaction in a repeatable manner and was thus selected for initial
inhibition trials. Although these trials did not detect inhibition of this protein-protein
interaction, the conserved alpha-helical regions involved in the nps7/nsp8
interface remain a promising avenue for targeted development of antivirals.