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Principal Investigator: Michael Grigg
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $454,297
Funding agency: National Institute of Allergy and Infectious Diseases
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiologic agent of COVID-19, emerged in 2019 as a public health emergency and global pandemic. SARS-CoV-2 is a linear, non-segmented, capped, polyadenylated positive-sense, single stranded RNA virus related to SARS-CoV and MERS-CoV, two other deadly respiratory coronaviruses that caused human epidemics in 2002 and 2012, respectively. Two principal ways that Beta-Coronaviruses evolve diversity is by accumulating spontaneous mutations (genetic drift) and by recombination (admixing genome segments of different ancestry). SARS-CoV (also known as beta-CoV) is a mosaic of alpha and gamma-CoV lineages and is thought to have evolved by recombination between mammalian-like and avian-like parent viruses. Likewise, surveillance studies of the MERS-CoV epidemic established that the origin of the 2015 human outbreak strain was generated by recombination among different MERS-CoV clade B lineages co-circulating in dromedary camels in Saudi Arabia in 2014. Our proposal will determine the genome-scale evolutionary dynamics of both SARS-CoV-2 and MERS-CoV by visualizing multiple evolutionary relationships to understand the role of recombination in the emergence and genetic origin of the current population genetic structure of SARS-CoV-2 and MERS-CoV. The Molecular Parasitology Section (LPD, NIAID) has extensive experience conducting population genomic studies and is vested in both the knowledge and techniques to adapt the current methodology we use to study haploid eukaryotic genomes to haploid, non-segmented RNA virus genomes. Furthermore, we are pursuing an active vaccine program by co-expressing parasite surface antigens in enveloped virus-like particles (eVLPs) as part of our oral and intra-nasal vaccine effort to generate protective, neutralizing antibodies and strong mucosal immunity against protozoan parasites. Our plan is to adapt (effectively re-purpose) this methodology to generate immunity against SARS-CoV-2 spike and N proteins. Our vaccine initiative represents one of the few programs involved in generating an effective oral or intra-nasal vaccine against respiratory viruses that cause COVID-19. Finally, the Spike protein of SARS-CoV-2 is essential for viral entry. The N-terminal domain (NTD) of S induces a class of neutralizing antibodies that fail to block ACE-2 receptor engagement but rather, lock the S protein in its pre-fusion state. In the first 100 amino acids of the S NTD we have identified two highly conserved sequence motifs: a PPXY binding motif for Nedd4-ubiquitin ligases at amino acid position 25, and three N-glycosylation sites that utilize the canonical NXT sequence at amino acid positions 17, 61 and 74. Structural modelling of this region suggests that the N-glycosylation sites form a shield that controls access of the ubiquitin ligase(s) to PPXY which impacts virus fusion and entry kinetics. This region is also a hotspot for recombination and evolutionary selection among all naturally emerging SARS-CoV-2 lineages, which may underpin its functional significance. Our aim is to determine the role of S ubiquitination in virus entry into host cells, which has implications for antibody responses and evolutionary selection of this region of the S protein.
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