Many prokaryotic cells are encapsulated by a surface layer (S-layer) that consists of repeating units of S-layer proteins (SLPs). S-layers protect cells from the outside, provide mechanical stability, and also play roles in pathogenicity. In situ structural information on this highly abundant class of proteins is scarce, therefore atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified C. crescentus’ sole SLP RsaA we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. Next, we solved a 7.4 Å structure of the S-layer through electron cryotomography (cryo-ET) and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the cryo-ET map, resulting in a pseudo-atomic level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with cryo-ET and sub-nanometre resolution sub-tomogram averaging. The cellular S-layer atomic structure shows that the S-layer is porous, with the largest gap dimension being 27 Å, and is stabilised by multiple Ca2+ ions bound near interfaces.

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup. IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

Human noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Previous structural analysis of norovirus VLPs showed that the capsid has a T=3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM VLP structures of two GII.4 variants, termed CHDC-1974 and NSW-2012. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T=4 icosahedral symmetry. We found that the T=4 VLPs showed several structural differences to the T=3 VLPs. The T=4 particles assemble from 240 copies of VP1 that adopt four quasi-equivalent conformations (A, B, C, and D) that form two distinct dimers, A/B and C/D. The T=4 protruding domains were elevated ~21-Å off the capsid shell, which was ~7-Å more than the previously studied GII.10 T=3 VLPs. A small cavity and flap-like structure at the icosahedral twofold axis disrupted the contiguous T=4 shell, a consequence of the Dsubunit S-domains having smaller contact interfaces with neighboring dimers.Overall, our findings that old and new GII.4 VP1 sequences assemble T=4 VLPs might have implications for the design of potential future vaccines.

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.

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18 19 Human noroviruses are a leading cause of acute gastroenteritis, yet there are still no 20 vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in 21 insect cells typically results in the formation of virus-like particles (VLPs) that are 22 IMPORTANCE 39 40The discovery that the GII.4 VLPs have a T=4 symmetry is of significance, since this 41 represents the first known T=4 calicivirus structure. Interestingly, the GII.4 2012 42 variant shares 96% amino acid identity with a current GII.4 VLP vaccine candidate 43 sequence, which suggests that this vaccine might also have a T=4 symmetry. Our 44 previous results with these GII.4 VLPs showed functional binding properties to 45 antibodies and Nanobodies that were raised against T=3 (GII.10) VLPs. This suggests 46 that the T=4 VLPs were antigenically comparable to T=3 particles, despite the 47 (EM). The VLPs were diluted 1:30 in distilled water and applied to EM grids. The 130 grids were washed with distilled water, stained with 0.75% uranyl acetate, and the 131 excess uranyl acetate was removed with filter paper. Virion samples were applied to 132 EM grids, washed with water, fixed with 4% glutaraldehyde, and then stained as 133 above. EM images were acquired on a Zeiss 910 electron microscope at 50,000× 134 magnification. 135

Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics.