Protein engineering of invertase for enhancing yeast dough fermentation under high-sucrose conditions.

During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.

Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimer in Saccharomyces cerevisiae.

In an effort to control the outbreak of the African Swine Fever Virus (ASFV), there is an urgent need to develop an effective method to prevent the pandemic, including vaccines and diagnostic methods. The major capsid protein of ASFV p72 (B646L), which forms a trimer with each monomer adopting a double jelly roll fold, is the main component of the virus particle and major antigen of ASFV. Thus, the p72 protein may be considered an antigen candidate for vaccine and diagnostic development. However, the development of ASFV p72 trimer for the industry application, including veterinary usage, faces unavoidable challenges: firstly, the low cost of the antigen production is required in vaccine and diagnostic application; and, secondly, whether produced antigen folds in its native conformation. Here, based on the information provided by the atomic structure of p72, we have successfully performed rational mutagenesis on p72 trimers and expressed it in Saccharomyces cerevisiae with high yields. The cryo-EM structure of recombinant expressed p72 trimer is determined at 4.18 Å in resolution. The correlation coefficient between this structure and the ASFV virus structure is 0.77, suggesting a highly similar fold of this trimer with the native protein on the virus particle.

Structure of Senecavirus A 3C Protease Revealed the Cleavage Pattern of 3C Protease in Picornaviruses.

Senecavirus A (SVA) is an emerging picornavirus infecting porcine of all age groups and causing foot and mouth disease (FMD)-like symptoms. One of its key enzymes is the 3C protease (3Cpro), which is similar to other picornaviruses and essential for virus maturation by controlling polyprotein cleavage and RNA replication. In this study, we reported the crystal structure of SVA 3Cpro at a resolution of 1.9 Å and a thorough structural comparison against all published picornavirus 3Cpro structures. Using statistical and graphical visualization techniques, we also investigated the sequence specificity of the 3Cpro. The structure revealed that SVA 3Cpro adopted a typical chymotrypsin-like fold with the S1 subsite as the most conservative site among picornavirus 3Cpro. The surface loop, A1-B1 hairpin, adopted a novel conformation in SVA 3Cpro and formed a positively charged protrusion around S' subsites. Correspondingly, SVA scissile bonds preferred Asp rather than neutral amino acids at P3' and P4'. Moreover, SVA 3Cpro showed a wide range tolerance to P4 residue volume (acceptable range: 67 Å3 to 141 Å3), such as aromatic side chain, in contrast to other picornaviruses. In summary, our results provided valuable information for understanding the cleavage pattern of 3Cpro. IMPORTANCE Picornaviridae is a group of RNA viruses that harm both humans and livestock. 3Cpro is an essential enzyme for picornavirus maturation, which makes it a promising target for antiviral drug development and a critical component for virus-like particle (VLP) production. However, the current challenge in the development of antiviral drugs and VLP vaccines includes the limited knowledge of how subsite structure determines the 3Cpro cleavage pattern. Thus, an extensive comparative study of various picornaviral 3Cpro was required. Here, we showed the 1.9 Å crystal structure of SVA 3Cpro. The structure revealed similarities and differences in the substrate-binding groove among picornaviruses, providing new insights into the development of inhibitors and VLP.

Purification-induced damage to calicivirus particles at near-atomic resolution.

The purification of virus particles is an essential process for the manufacture of vaccines. However, the application of different purification processes may affect the quality of the virus particles, such as structural integrity and homogeneity, which may further influence the infectivity and immunogenicity of the purified virus. In this study, we took Feline calicivirus (FCV), a common natural pathogen in cats belonging to Caliciviridae, as a research model. By using cryo-electron microscopy (cryo-EM), we incorporated the 3D classification process as a virus flexibility evaluation system. Cryo-EM images of virus particles resulting from different purification processes were compared at near-atomic resolution. The results indicated that molecular sieving purification will impact the stability of P-domains through increasing flexibility as determined by the evaluation system, which can be extended to assess the purification effect on the entire particle. This evaluation process can be further applied to all non-enveloped viruses.

Nucleocapsid mutations R203K/G204R increase the infectivity, fitness, and virulence of SARS-CoV-2.

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.

Rapid Spread of Mutant Alleles in Worldwide SARS-CoV-2 Strains Revealed by Genome-Wide Single Nucleotide Polymorphism and Variation Analysis.

The novel coronavirus (SARS-CoV-2) has become a pandemic and is threatening human health globally. Here, we report nine newly evolved SARS-CoV-2 single nucleotide polymorphism (SNP) alleles those underwent a rapid increase (seven cases) or decrease (two cases) in their frequency for 30-80% in the initial four months, which are further confirmed by intrahost single nucleotide variation analysis using raw sequence data including 8,217 samples. The nine SNPs are mostly (8/9) located in the coding region and are mainly (6/9) nonsynonymous substitutions. The nine SNPs show a complete linkage in SNP pairs and belong to three different linkage groups, named LG_1 to LG_3. Analyses in population genetics show signatures of adaptive selection toward the mutants in LG_1, but no signal of selection for LG_2. Population genetic analysis results on LG_3 show geological differentiation. Analyses on geographic COVID-19 cases and published clinical data provide evidence that the mutants in LG_1 and LG_3 benefit virus replication and those in LG_1 have a positive correlation with the disease severity in COVID-19-infected patients. The mutants in LG_2 show a bias toward mildness of the disease based on available public clinical data. Our findings may be instructive for epidemiological surveys and disease control of COVID-19 in the future.

Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72.

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.

A database resource and online analysis tools for coronaviruses on a historical and global scale.

The recent outbreak of COVID-19 caused by a new zoonotic origin coronavirus (SARS-CoV-2 or 2019-nCoV) has sound the alarm for the potential spread of epidemic coronavirus crossing species. With the urgent needs to assist disease control and to provide invaluable scientific information, we developed the coronavirus database (CoVdb), an online genomic, proteomic and evolutionary analysis platform. CoVdb has brought together genomes of more than 5000 coronavirus strains, which were collected from 1941 to 2020, in more than 60 countries and in hosts belonging to more than 30 species, ranging from fish to human. CoVdb presents comprehensive genomic information, such as gene function, subcellular localization, topology and protein structure. To facilitate coronavirus research, CoVdb also provides flexible search approaches and online tools to view and analyze protein structure, to perform multiple alignments, to automatically build phylogenetic trees and to carry on evolutionary analyses. CoVdb can be accessed freely at http://covdb.popgenetics.net. Hopefully, it will accelerate the progress to develop medicines or vaccines to control the pandemic of COVID-19.

Genomic recombination events may reveal the evolution of coronavirus and the origin of SARS-CoV-2.

To trace the evolution of coronaviruses and reveal the possible origin of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19), we collected and thoroughly analyzed 29,452 publicly available coronavirus genomes, including 26,312 genomes of SARS-CoV-2 strains. We observed coronavirus recombination events among different hosts including 3 independent recombination events with statistical significance between some isolates from humans, bats and pangolins. Consistent with previous records, we also detected putative recombination between strains similar or related to Bat-CoV-RaTG13 and Pangolin-CoV-2019. The putative recombination region is located inside the receptor-binding domain (RBD) of the spike glycoprotein (S protein), which may represent the origin of SARS-CoV-2. Population genetic analyses provide estimates suggesting that the putative introduced DNA within the RBD is undergoing directional evolution. This may result in the adaptation of the virus to hosts. Unsurprisingly, we found that the putative recombination region in S protein was highly diverse among strains from bats. Bats harbor numerous coronavirus subclades that frequently participate in recombination events with human coronavirus. Therefore, bats may provide a pool of genetic diversity for the origin of SARS-CoV-2.