Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus

Abstract

Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg2+ or Ca2+. Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties. IMPORTANCE This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.

Research progress and applications of nanobody in human infectious diseases

“…There are many different steps during the life cycle of pathogens in the body, including cell attachment and entry, replication and regulation, transcription and translation, and virion assembly, offering diverse targets (Koromyslova et al, 2020;Tsukuda and Watashi, 2020). Pathogens have low homology with humans, exhibiting a variety of attractive epitopes.…”

“…Nanobodies that bind at a dimeric interface on the lower side of the P dimer disrupt a structural change in the capsid associated with binding cofactors. Furthermore, nanobodies induce conformational rearrangement of several P domain loops, leading to particle aggregation and interference at the HBGA binding pocket (Koromyslova and Hausman, 2018;Koromyslova et al, 2020). Kerstin Ruoff et al also demonstrated that nanobodies have different targets and inhibit norovirus infection through various mechanisms.…”

NanoNet: Rapid end-to-end nanobody modeling by deep learning at sub angstrom resolution

“…Here we tested whether the accuracy of NanoNet Nb models is sufficient to enable sampling of the correct orientation. Docking was applied for 17 Nbs (7 Nbs that interact with SARS-CoV-2 spike RBD [20][21][22][23][24] , 2 Nbs that bind SARS-CoV-2 N protein 25 , 1 Nb binding to SARS-CoV-1 spike RBD, 1 Nb binding to the Ebola RNA methyltransferase 26 , and 4 MNV capsid protein P-domain Nbs 27 ). In addition, we docked two high affinity SARS-CoV-2 RBD Nbs (Nb21, Nb105) that bind to different epitopes 28 .…”

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid

“…This observation is surprising in the context of the previous literature, which would have better supported a role for specific P2 amino acids in mediating altered tropism and increased virulence. However, recent reports have suggested that norovirus capsid structures may be highly dynamic and that the P1 domain may govern this flexibility by regulating interactions with the shell domain [58][59][60][61][62]. We hypothesize that residue 514 may be involved in this flexibility, as several amino acids with which it is predicted to interact make up a portion of this flexible linker, in the capsids of both MNoV S7 (which possesses F514) and CW3 (which possesses I514) (S9 Fig) . F/I514 may influence this linker to adopt either the "expanded" or "closed" conformation, changing the capsid structure and altering its ability to interact with receptors and/ or the immune system.…”