UBC Theses and Dissertations
Molecular mechanism of a novel nuclear entry pathway used by the parvovirus minute virus of mice Yang, Shuang
Being non-pathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses that have great potential as anti-cancer agents. Since these viruses replicate in the nucleus of the host cell, they have to overcome various cellular barriers, including the nuclear envelope (NE), before reaching the nucleus. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the NE and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. Therefore, this thesis uses MVM as a model to identify the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays and mutagenesis, we identified a functional internal N-myristoylation site starting with glycine (G) at position 78 in the unique portion of the viral structural protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this N-myristoylation site or with purified virions, we found that tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying that this cleavage exposes G78 at the N-terminus of VP1u for N-myristoylation. Subsequent experiments were conducted in MVM-infected cells or using an NE permeabilization assay with inhibitors of N-myristoylation, chymotrypsin-like proteases, or the chymotrypsin-like activity of the proteasome. These results provide further evidence that protein N-myristoylation and a chymotrypsin-like activity, which may come from cellular proteasomes, are essential for MVM-induced NE disruption. In addition to MVM VP1, we investigated the role of proteolytic cleavage at the N-terminus of VP2 (the other structural protein of MVM) in viral nuclear entry. By generating and studying MVM with cleaved VP2 N-termini, we demonstrated that the VP2 N-terminus cleavage during early infection steps of MVM makes the virion more infectious than the wild-type one by enhancing its ability to disrupt the NE during viral nuclear entry. Similar to the wild-type MVM, the VP2 N-termini-cleaved virion also requires protein N-myristoylation and proteasomal processing to induce NE disruption. These findings on the mechanism of MVM nuclear entry would deepen our understanding of the basic biology of PVs and contribute to the development of PVs-based therapeutics.
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