The binding and structural basis of fox ACE2 to RBDs from different sarbecoviruses.

Foxes are susceptible to SARS-CoV-2 in laboratory settings, and there have also been reports of natural infections of both SARS-CoV and SARS-CoV-2 in foxes. In this study, we assessed the binding capacities of fox ACE2 to important sarbecoviruses, including SARS-CoV, SARS-CoV-2, and animal-origin SARS-CoV-2 related viruses. Our findings demonstrated that fox ACE2 exhibits broad binding capabilities to receptor-binding domains (RBDs) of sarbecoviruses. We further determined the cryo-EM structures of fox ACE2 complexed with RBDs of SARS-CoV, SARS-CoV-2 prototype (PT), and Omicron BF.7. Through structural analysis, we identified that the K417 mutation can weaken the ability of SARS-CoV-2 sub-variants to bind to fox ACE2, thereby reducing the susceptibility of foxes to SARS-CoV-2 sub-variants. In addition, the Y498 residue in the SARS-CoV RBD plays a crucial role in forming a vital cation-π interaction with K353 in the fox ACE2 receptor. This interaction is the primary determinant for the higher affinity of the SARS-CoV RBD compared to that of the SARS-CoV-2 PT RBD. These results indicate that foxes serve as potential hosts for numerous sarbecoviruses, highlighting the critical importance of surveillance efforts.

Structural basis of increased binding affinities of spikes from SARS-CoV-2 Omicron variants to rabbit and hare ACE2s reveals the expanding host tendency.

The potential host range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been expanding alongside its evolution during the pandemic, with rabbits and hares being considered important potential hosts, supported by a report of rabbit sero-prevalence in nature. We measured the binding affinities of rabbit and hare angiotensin-converting enzyme 2 (ACE2) with receptor-binding domains (RBDs) from SARS-CoV, SARS-CoV-2, and its variants and found that rabbit and hare ACE2s had broad variant tropism, with significantly enhanced affinities to Omicron BA.4/5 and its subsequent-emerged sub-variants (>10 fold). The structures of rabbit ACE2 complexed with either SARS-CoV-2 prototype (PT) or Omicron BA.4/5 spike (S) proteins were determined, thereby unveiling the importance of rabbit ACE2 Q34 in RBD-interaction and elucidating the molecular basis of the enhanced binding with Omicron BA.4/5 RBD. These results address the highly enhanced risk of rabbits infecting SARS-CoV-2 Omicron sub-variants and the importance of constant surveillance.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the globe and caused immense health and economic damage. SARS-CoV-2 has demonstrated a broad host range, indicating a high risk of interspecies transmission and adaptive mutation. Therefore, constant monitoring for potential hosts is of immense importance. In this study, we found that Omicron BA.4/5 and subsequent-emerged sub-variants exhibited enhanced binding to both rabbit and hare angiotensin-converting enzyme 2 (ACE2), and we elucidated the structural mechanism of their recognition. From the structure, we found that Q34, a unique residue of rabbit ACE2 compared to other ACE2 orthologs, plays an important role in ACE2 recognition. These results address the probability of rabbits/hares being potential hosts of SARS-CoV-2 and broaden our knowledge regarding the molecular mechanism of SARS-CoV-2 interspecies transmission.

Cross-species recognition of bat coronavirus RsYN04 and cross-reaction of SARS-CoV-2 antibodies against the virus.

Following the outbreak of coronavirus disease 2019 (COVID-19), several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related coronaviruses have been discovered. Previous research has identified a novel lineage of SARS-CoV-2-related CoVs in bats, including RsYN04, which recognizes human angiotensin-converting enzyme 2 (ACE2) and thus poses a potential threat to humans. Here, we screened the binding of the RsYN04 receptor-binding domain (RBD) to ACE2 orthologs from 52 animal species and found that the virus showed a narrower ACE2-binding spectrum than SARS-CoV-2. However, the presence of the T484W mutation in the RsYN04 RBD broadened its range. We also evaluated 44 SARS-CoV-2 antibodies targeting seven epitope communities in the SARS-CoV-2 RBD, together with serum obtained from COVID-19 convalescents and vaccinees, to determine their cross-reaction against RsYN04. Results showed that no antibodies, except for the RBD-6 and RBD-7 classes, bound to the RsYN04 RBD, indicating substantial immune differences from SARS-CoV-2. Furthermore, the structure of the RsYN04 RBD in complex with cross-reactive antibody S43 in RBD-7 revealed a potently broad epitope for the development of therapeutics and vaccines. Our findings suggest RsYN04 and other viruses belonging to the same clade have the potential to infect several species, including humans, highlighting the necessity for viral surveillance and development of broad anti-coronavirus countermeasures.

Structural basis for receptor binding and broader interspecies receptor recognition of currently circulating Omicron sub-variants.

Multiple SARS-CoV-2 Omicron sub-variants, such as BA.2, BA.2.12.1, BA.4, and BA.5, emerge one after another. BA.5 has become the dominant strain worldwide. Additionally, BA.2.75 is significantly increasing in some countries. Exploring their receptor binding and interspecies transmission risk is urgently needed. Herein, we examine the binding capacities of human and other 28 animal ACE2 orthologs covering nine orders towards S proteins of these sub-variants. The binding affinities between hACE2 and these sub-variants remain in the range as that of previous variants of concerns (VOCs) or interests (VOIs). Notably, R493Q reverse mutation enhances the bindings towards ACE2s from humans and many animals closely related to human life, suggesting an increased risk of cross-species transmission. Structures of S/hACE2 or RBD/hACE2 complexes for these sub-variants and BA.2 S binding to ACE2 of mouse, rat or golden hamster are determined to reveal the molecular basis for receptor binding and broader interspecies recognition.

Expression of an extremely acidic beta-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence.

BACKGROUND: Alicyclobacillus sp. A4 is thermoacidophilic and produces many glycoside hydrolases. An extremely acidic beta-1,4-glucanase (CelA4) has been isolated from Alicyclobacillus sp. A4 and purified. This glucanase with a molecular mass of 48.6 kDa decreases the viscosity of barley-soybean feed under simulated gastric conditions. Therefore, it has the potential to improve the nutrient bioavailability of pig feed. For the study reported herein, the full-length gene, CelA4, of this glucanase (CelA4) was identified using the sequences of six peptides and cloned from strain A4. The gene fragment (CelA4F) encoding the mature protein was expressed in Pichia pastoris. Sequence truncation and glycosylation were found for recombinant CelA4F, both of which affected the expression efficiency. The physical properties of various forms of CelA4 as they affected enzymatic activity were characterized. RESULTS: We located the full-length 2,148-bp gene for CelA4 (CelA4) in the genome of Alicyclobacillus sp. A4. CelA4 encodes a 715-residue polypeptide with a calculated molecular mass of 71.64 kDa, including an N-terminal signal peptide (residues 1-39), a catalytic domain (residues 39-497), and a C-terminal threonine-rich region (residues 498-715). Its deduced amino acid sequence and that of an Alicyclobacillus acidocaldarius endo-beta-1,4-glucanase were identical at 44% of the residue positions. When the experimental molecular mass of CelA4F--a recombinant protein designed to mimic the CelA4 sequence lacking the N-terminal signal peptide that had been expressed in Pichia pastoris--was compared with its hypothetical molecular mass, it was apparent that CelA4F was truncated, possibly at residue 497. An artificially truncated gene fragment (CelA4T) without C-terminal threonine-rich region was expressed in P. pastoris, and the expression efficiency of CelA4T was substantially greater than that of CelA4F. Purified CelA4F and CelA4T had similar molecular masses (~60 kDa) and enzymatic properties (optimum pH, 3.4; optimum temperature, 60 degrees C); they were relatively stable between pH 1.2 and 8.2 at 70 degrees C and resistant to acidic and neutral proteases. However, their molecular masses and thermostabilities differed from those of CelA4 isolated from Alicyclobacillus sp. A4. A deglycosylated form of CelA4 (CelA4D) had properties similar to that of CelA4 except that it was thermoliable at 60 degrees C. CONCLUSIONS: Truncation during expression of CelA4F or artificial truncation of its gene--both of which produced a form of CelA4 lacking a threonine-rich region that includes a putative linker--increased the level of enzyme produced in comparison with that produced by cultivation of Alicyclobacillus sp. A4. Glycosylation increased the thermostability of CelA4. Of the four forms of CelA4 studied, CelA4T was produced in highest yield and had the most favorable physical properties; therefore, it has potential for use in the feed industry.

A novel family 9 beta-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry.

An endo-beta-1,3(4)-glucanase gene, Agl9A, was cloned from Alicyclobacillus sp. A4 and expressed in Pichia pastoris. Its deduced amino acid sequence shared the highest identity (48%) with an endo-beta-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A exhibited relatively wide substrate specificity, including lichenan (109%), barley beta-glucan (100%), CMC-Na (15.02%), and laminarin (6.19%). The optimal conditions for Agl9A activity were pH 5.8 and 55 degrees C. The enzyme was stable over a broad pH range (>60% activity retained after 1-h incubation at pH 3.8-11.2) and at 60 degrees C (>70% activity retained after 1-h incubation). Agl9A was highly resistant to various neutral proteases (e.g., trypsin, alpha-chymotrypsin, and collagenase) and Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A (20 U/ml) or a commercial xylanase (200 U/ml) reduced the filtration rate (26.71% and 20.21%, respectively) and viscosity (6.12% and 4.78%, respectively); furthermore, combined use of Agl9A (10 U/ml) and the xylanase (100 U/ml) even more effectively reduced the filtration rate (31.73%) and viscosity (8.79%). These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.