Transgenic expression of artificial microRNA targeting soybean mosaic virus P1 gene confers virus resistance in plant.

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

X-ray Crystal Structures of the Influenza M2 Proton Channel Drug-Resistant V27A Mutant Bound to a Spiro-Adamantyl Amine Inhibitor Reveal the Mechanism of Adamantane Resistance.

The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex.

A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa.

The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common ß-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

The Characterization of the Tobacco-Derived Wild Tomato Mosaic Virus by Employing Its Infectious DNA Clone.

Viral diseases of cultivated crops are often caused by virus spillover from wild plants. Tobacco (N. tabacum) is an important economic crop grown globally. The viral pathogens of tobacco are traditional major subjects in virology studies and key considerations in tobacco breeding practices. A positive-strand RNA virus, wild tomato mosaic virus (WTMV), belonging to the genus potyvirus in the family potyviridae was recently found to infect tobacco in China. In this study, diseased tobacco leaf samples were collected in the Henan Province of China during 2020-2021. Several samples from different locations were identified as WTMV positive. An infectious DNA clone was constructed based on one of the WTMV isolates. By using this clone, we found that WTMV from tobacco could establish infections on natural reservoir hosts, demonstrating a possible route of WTMV spillover and overwintering in the tobacco field. Furthermore, the WTMV infection was found to be accompanied by other tobacco viruses in the field. The co-inoculation experiments indicate the superinfection exclusion (SIE) between WTMV and other potyvirus species that infect tobacco. Overall, our work reveals novel aspects of WTMV evolution and infection in tobacco and provides an important tool for further studies of WTMV.

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2.

SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants.

SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 µg mL-1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 µg mL-1 for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 µg mL-1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication.

BACKGROUND: Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. RESULTS: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. CONCLUSIONS: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

Synergistic antifibrotic effect of verapamil and interferon-gamma in rats: partially based on enhanced verapamil oral bioavailability.

OBJECTIVE: The objective of this study was to investigate the synergistic antifibrotic effect of verapamil and interferon-gamma (IFN-gamma) on rat liver fibrosis and its potential pharmacokinetic-based mechanism. METHODS: Rat liver fibrosis model was successfully established, and both the therapeutic effects and pharmacokinetic parameters of verapamil were evaluated after the administration of verapamil with or without IFN-gamma. The activities of cytochrome P450 3A (CYP3A) and the expression of multidrug resistance (Mdr) mRNA were measured in liver and small intestine. RESULTS: The results showed the synergistic antifibrotic effect of verapamil and IFN-gamma in rat liver fibrosis, in terms of decreased serum L-alanine aminotransferase activity and liver hydroxyproline content and improved liver histopathology, when compared with rats treated with verapamil or IFN-gamma alone. Meanwhile, the area under the curve of verapamil increased significantly after single administration of verapamil and IFN-gamma and the concentration of verapamil in plasma increased, but the metabolite : parent ratio of verapamil decreased after consecutive administrations of verapamil and IFN-gamma. Furthermore, the activities of CYP3A in both the liver and the small intestine and the expression of Mdr in small intestine decreased in rats treated with verapamil and IFN-gamma. CONCLUSION: All these results indicated that the combination of verapamil and IFN-gamma exerts a synergistic antifibrotic effect on rat liver fibrosis. The mechanism was partially based on the enhanced oral bioavailability of verapamil by increasing the intestinal absorption as well as reducing the first-pass metabolism, through inhibition of CYP3A activity and P-glycoprotein expression by IFN-gamma

Distribution of signal transducer and activator of transcription 6 gene G2964A polymorphism in Chinese patients with ulcerative colitis.

BACKGROUND AND AIMS: The signal transducer and activator of transcription 6 (STAT6) gene is located on chromosome 12q13.3-14.1 just within the IBD2 region and is a key transcription factor involved in interleukin (IL)-4 and IL-13-mediated Th2 response. The aim of the present study was to determine distribution of the STAT6 gene polymorphism in Chinese patients with ulcerative colitis. METHODS: The G2964A polymorphism in the 3' untranslated region of the STAT6 gene was studied in 84 unrelated Chinese patients with ulcerative colitis and 176 healthy controls by PCR and the amplification created restriction site method. The results were then compared with those from a Dutch study published previously. RESULT: Significant differences in genotype and allele frequencies of the STAT6 G2964A polymorphism were not found between patients with ulcerative colitis and healthy controls. Subgroups of the patients with ulcerative colitis classified according to the age at onset, sex and location of disease did not differ significantly in the distribution of this polymorphism. However, the genotypes (P < 0.0001, chi-squared = 75.332) and allele frequencies (P < 0.0001, odds ratio = 4.298, 95% confidence interval = 3.070-6.018) were significantly different between the Chinese and Dutch populations. CONCLUSION: The STAT6 G2964A polymorphism is not involved in the genetic susceptibility to ulcerative colitis in Chinese patients.

MHC class I chain-related gene A-A5.1 allele is associated with ulcerative colitis in Chinese population.

The human MHC class I chain-related gene A (MICA) plays a role in regulating protective responses by intestinal epithelial Vdelta1 gamma delta T cells and the polymorphism of MICA were reported to be related to several autoimmune diseases. The present study aimed to investigate the association of the microsatellite polymorphisms of TM region of MICA gene with the susceptibility to ulcerative colitis (UC) in Chinese population. The microsatellite polymorphisms of the MICA were genotyped in unrelated 86 Chinese patients with UC and 172 ethnically matched healthy controls by a semiautomatic fluorenscently labelled PCR method. All the subjects were the Chinese with Han nationality. The frequency of MICA-A5.1 homozygous genotype and A5.1 allele were significantly increased in UC patients compared with healthy controls (22.1%versus 7%, P = 0.0009, Pc = 0.0126, OR = 3.781, 95%CI: 1.738-8.225 and 30.2%versus 17.4%, P = 0.0014, Pc = 0.007, OR = 2.051, 95%CI: 1.336-3.148, respectively). Adjusted the effects of gender and age at onset, MICA-A5.1 homozygous genotype and A5.1 allele were also increased in the UC patients. Moreover MICA-A5.1 allele was significantly increased in frequency in the female UC patients (38.2%versus 21.0%, P = 0.0095, Pc = 0.0475, OR = 2.326, 95%CI: 1.234-4.382). Logistic regression analysis also revealed that gender was independently associated with UC patients carried MICA-A5.1 allele (P = 0.046, OR (male) = 0.511, 95% CI: 0.264-0.987). Although the UC patients with extensive colitis (32.5%versus 17.4% in the healthy controls, P = 0.005, Pc = 0.025) and the UC patients with extraintestinal manifestations (36%versus 17.4% in the healthy controls, P = 0.0039, Pc = 0.0195) were more likely to carry the MICA-A5.1 allele, EIMs was associated with extent of disease (P < 0.0001, OR (with EIMs) = 3.511, 95% CI 1.747-7.056) and MICA-A5.1 allele was not associated with UC patients with extensive colitis or with EIMs in the logistic regression analysis. Therefore, the MICA-A5.1 homozygous genotype and A5.1 allele were closely associated with UC and the MICA-A5.1 allele was positively associated with the female UC patients in Chinese population.