Anna D. Koromyslova

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 analysed 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 analysed the inhibition mechanisms using X-ray crystallography, cryo-EM, and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, the 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 co-factors (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 post-receptor 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 analysed 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 co-factor 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 co-factor/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.

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 analysed 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 analysed the inhibition mechanisms using X-ray crystallography, cryo-EM, and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, the 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 co-factors (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 post-receptor attachment stage. Altogether, our new data show that Nanobody-based inhibition could occur by blocking functional and structural capsid properties.AUTHOR SUMMARY: This research discovered and analysed 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 co-factor 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 co-factor/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.

Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Å resolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five β-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.

The emergence of new epidemic GII.4 norovirus variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between the 1974 and 2012 GII.4 variants, whereas these variants showed various levels of reactivity against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket, blocked norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4 1974 isolate but was able to neutralize the newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggest that the amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for infection.

Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The GII.4 persistence is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolate (1974) and a more recent epidemic GII.4 variant (2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities, yet similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivities with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and haddirect competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus VLPs from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunityand impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.

Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. We determined the binding sites of six novel human norovirus specific Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies (Nano-4, Nano-26, Nano-27, and Nano-42) bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding region is highly variable and inherently suffers from lack of conservation among the genetically diverse genotypes. Instead, we showed that the capsid contained other highly susceptible regions that could be targeted for virus inhibition.