Conformations of membrane human immunodeficiency virus (HIV-1) envelope glycoproteins solubilized in Amphipol A18 lipid-nanodiscs.

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

A novel class of xylanases specifically degrade marine red algal ß1,3/1,4-mixed-linkage xylan.

Xylans are polysaccharides composed of xylose and include ß1,4-xylan, ß1,3-xylan, and ß1,3/1,4-mixed-linkage xylan (MLX). MLX is widely present in marine red algae and constitutes a significant organic carbon in the ocean. Xylanases are hydrolase enzymes that play an important role in xylan degradation. While a variety of ß1,4-xylanases and ß1,3-xylanases involved in the degradation of ß1,4-xylan and ß1,3-xylan have been reported, no specific enzyme has yet been identified that degrades MLX. Herein, we report the characterization of a new MLX-specific xylanase from the marine bacterium Polaribacter sp. Q13 which utilizes MLX for growth. The bacterium secretes xylanases to degrade MLX, among which is Xyn26A, an MLX-specific xylanase that shows low sequence similarities (<27%) to ß1,3-xylanases in the glycoside hydrolase family 26 (GH26). We show that Xyn26A attacks MLX precisely at ß1,4-linkages, following a ß1,3-linkage toward the reducing end. We confirm that Xyn26A and its homologs have the same specificity and mode of action on MLX, and thus represent a new xylanase group which we term as MLXases. We further solved the structure of a representative MLXase, AlXyn26A. Structural and biochemical analyses revealed that the specificity of MLXases depends critically on a precisely positioned ß1,3-linkage at the -2/-1 subsite. Compared to the GH26 ß1,3-xylanases, we found MLXases have evolved a tunnel-shaped cavity that is fine-tuned to specifically recognize and hydrolyze MLX. Overall, this study offers a foremost insight into MLXases, shedding light on the biochemical mechanism of bacterial degradation of MLX.

Structural and molecular basis for urea recognition by Prochlorococcus.

Nitrogen (N) is an essential element for microbial growth and metabolism. The growth and reproduction of microorganisms in more than 75% of areas of the ocean are limited by N. Prochlorococcus is numerically the most abundant photosynthetic organism on the planet. Urea is an important and efficient N source for Prochlorococcus. However, how Prochlorococcus recognizes and absorbs urea still remains unclear. Prochlorococcus marinus MIT 9313, a typical Cyanobacteria, contains an ABC-type transporter, UrtABCDE, which may account for the transport of urea. Here, we heterologously expressed and purified UrtA, the substrate-binding protein of UrtABCDE, detected its binding affinity toward urea, and further determined the crystal structure of the UrtA/urea complex. Molecular dynamics simulations indicated that UrtA can alternate between "open" and "closed" states for urea binding. Based on structural and biochemical analyses, the molecular mechanism for urea recognition and binding was proposed. When a urea molecule is bound, UrtA undergoes a state change from open to closed surrounding the urea molecule, and the urea molecule is further stabilized by the hydrogen bonds supported by the conserved residues around it. Moreover, bioinformatics analysis showed that ABC-type urea transporters are widespread in bacteria and probably share similar urea recognition and binding mechanisms as UrtA from P. marinus MIT 9313. Our study provides a better understanding of urea absorption and utilization in marine bacteria.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

Effects of plant taxonomic position on soil nematode communities in Antarctica.

Antarctica terrestrial ecosystems are facing the most threats from global climate change, which is altering plant composition greatly. These transformations may cause major reshuffling of soil community composition, including functional traits and diversity, and therefore affect ecosystem processes in Antarctica. We used high-throughput sequencing analysis to investigate soil nematodes under 3 dominant plant functional groups (lichens, mosses, and vascular plants) and bare ground in the Antarctic region. We calculated functional diversity of nematodes based on their diet, life histories, and body mass with kernel density n-dimensional hypervolumes. We also calculated taxonomic and functional beta diversity of the nematode communities based on Jaccard dissimilarity. The presence of plants had no significant effect on the taxonomic richness of nematodes but significantly increased nematode functional richness. The presence of plants also significantly decreased taxonomic beta diversity (homogenization). Only mosses and vascular plants decreased nematode functional beta diversity, which was mostly due to a decreased effect of the richness difference component. The presence of plants also increased the effect of deterministic processes potentially because environmental filtering created conditions favorable to nematodes at low trophic levels with short life histories and small body size. Increasing plant cover in the Antarctic due to climate change may lead to increased diversity of nematode species that can use the scarce resources and nematode taxonomic and functional homogenization. In a future under climate change, community restructuring in the region is possible.

Efectos de la posición taxonómica de las plantas sobre las comunidades de nemátodos del suelo en la Antártida Resumen Los ecosistemas terrestres de la Antártida enfrentan las mayores amenazas del cambio climático global, que está alterando gravemente la composición de plantas. Estas transformaciones pueden provocar una reorganización importante de la composición de la comunidad del suelo, incluyendo atributos y diversidad funcionales, y por lo tanto afectar los procesos ecosistémicos en la Antártida. Utilizamos análisis de secuenciación de alto rendimiento para investigar nemátodos del suelo debajo de tres grupos funcionales de plantas dominantes (líquenes, musgos y plantas vasculares) y de suelo desnudo en la región de la Antártida. Calculamos la diversidad funcional de nemátodos con base en su dieta, historia de vida y masa corporal mediante hipervolúmenes n­dimensionales de densidad del núcleo. También calculamos la diversidad beta taxonómica y funcional de las comunidades de nemátodos con base en la disimilitud de Jacard. La presencia de plantas no tuvo efecto significativo sobre la riqueza taxonómica de nemátodos, pero incrementó su riqueza funcional significativamente. La presencia de plantas también disminuyó la diversidad beta taxonómica (homogenización) significativamente. Solo musgos y plantas vasculares disminuyeron la diversidad beta funcional de nemátodos, lo cual se debió principalmente a un menor efecto del componente de diferencia de riqueza. La presencia de plantas también incrementó el efecto de los procesos determinísticos posiblemente porque el filtrado ambiental creó condiciones favorables para los nemátodos de niveles tróficos inferiores con historias de vida corta y tamaño corporal pequeño. El incremento de la cobertura de plantas en la Antártida debido al cambio climático puede conducir a una mayor diversidad de especies de nemátodos que pueden utilizar los escasos recursos y a la homogenización taxonómica y funcional de los nemátodos. En un futuro bajo el cambio climático, es posible la reestructuración comunitaria en la región.

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein.

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Functional and Highly Cross-Linkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation.

Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

Comprehensive analysis of clinical indications and viral strain variants among patients infected with SARS-CoV-2 in Inner Mongolia, China.

Since the first appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, the virus is still evolving and mutating until now. In this study, we collected 6 throat swabs from patients who diagnosed with COVID-19 in Inner Mongolia, China, to understand the entry of multiple SARS-CoV-2 variants into Inner Mongolia and analyze the relationships between variants and clinical features observed in infected patients. In addition, we performed a combined analysis of clinical parameters associated with SARS-CoV-2 variants of interest, pedigree analysis, and detection of single-nucleotide polymorphisms. Our results showed that the clinical symptoms were generally mild although some patients demonstrated some degree of liver function abnormalities, and the SARS-CoV-2 strain was related to the Delta variant (B.1.617.2), AY.122 lineage. The epidemiological investigations and clinical manifestations confirmed that the variant exhibits strong transmission, a high viral load, and moderate clinical symptoms. SARS-CoV-2 has undergone extensive mutations in various hosts and countries. Timely monitoring of virus mutation can help to monitor the spread of infection and characterize the diversity of genomic variants, thus limiting future waves of SARS-CoV-2 infection.

Biochemical Insights into a Novel Family 2 Glycoside Hydrolase with Both ß-1,3-Galactosidase and ß-1,4-Galactosidase Activity from the Arctic.

A novel GH2 (glycoside hydrolase family 2) ß-galactosidase from Marinomonas sp. BSi20584 was successfully expressed in E. coli with a stable soluble form. The recombinant enzyme (rMaBGA) was purified to electrophoretic homogeneity and characterized extensively. The specific activity of purified rMaBGA was determined as 96.827 U mg-1 at 30 °C using ONPG (o-nitrophenyl-ß-D-galactopyranoside) as a substrate. The optimum pH and temperature of rMaBGA was measured as 7.0 and 50 °C, respectively. The activity of rMaBGA was significantly enhanced by some divalent cations including Zn2+, Mg2+ and Ni2+, but inhibited by EDTA, suggesting that some divalent cations might play important roles in the catalytic process of rMaBGA. Although the enzyme was derived from a cold-adapted strain, it still showed considerable stability against various physical and chemical elements. Moreover, rMaBGA exhibited activity both toward Galß-(1,3)-GlcNAc and Galß-(1,4)-GlcNAc, which is a relatively rare occurrence in GH2 ß-galactosidase. The results showed that two domains in the C-terminal region might be contributed to the ß-1,3-galactosidase activity of rMaBGA. On account of its fine features, this enzyme is a promising candidate for the industrial application of ß-galactosidase.

Fast Distributed Model Predictive Control Method for Active Suspension Systems.

In order to balance the performance index and computational efficiency of the active suspension control system, this paper offers a fast distributed model predictive control (DMPC) method based on multi-agents for the active suspension system. Firstly, a seven-degrees-of-freedom model of the vehicle is created. This study establishes a reduced-dimension vehicle model based on graph theory in accordance with its network topology and mutual coupling constraints. Then, for engineering applications, a multi-agent-based distributed model predictive control method of an active suspension system is presented. The partial differential equation of rolling optimization is solved by a radical basis function (RBF) neural network. It improves the computational efficiency of the algorithm on the premise of satisfying multi-objective optimization. Finally, the joint simulation of CarSim and Matlab/Simulink shows that the control system can greatly minimize the vertical acceleration, pitch acceleration, and roll acceleration of the vehicle body. In particular, under the steering condition, it can take into account the safety, comfort, and handling stability of the vehicle at the same time.

Coordinated Optimal Control of AFS and DYC for Four-Wheel Independent Drive Electric Vehicles Based on MAS Model.

The problem that it is difficult to balance vehicle stability and economy at the same time under the starting steering condition of a four-wheel independent drive electric vehicle (4WIDEV) is addressed. In this paper, we propose a coordinated optimal control method of AFS and DYC for a four-wheel independent drive electric vehicle based on the MAS model. Firstly, the angular velocity of the transverse pendulum at the center of mass and the lateral deflection angle of the center of mass are decoupled by vector transformation, and the two-degree-of-freedom eight-input model of the vehicle is transformed into four two-degree-of-freedom two-input models, and the reduced-dimensional system is regarded as four agents. Based on the hardware connection structure and communication topology of the four-wheel independent drive electric vehicle, the reduced-dimensional model of 4WIDEV AFS and DYC coordinated optimal control is established based on graph theory. Secondly, the deviation of the vehicle transverse swing angular velocity and mass lateral deflection angle from their ideal values is oriented by combining sliding mode variable structure control (SMC) with distributed model predictive control (DMPC). A discrete dynamic sliding mode surface function is proposed for the ith agent to improve the robustness of the system in response to parameter variations and disturbances. Considering the stability and economy of the ith agent, an active front wheel steering and drive torque optimization control method based on SMC and DMPC is proposed for engineering applications. Finally, a hardware-in-the-loop (HIL) test bench is built for experimental verification, and the results show that the steering angle is in the range of 0-5°, and the proposed method effectively weighs the system dynamic performance, computational efficiency, and the economy of the whole vehicle. Compared with the conventional centralized control method, the torque-solving speed is improved by 32.33 times, and the electrical consumption of the wheel motor is reduced by 16.6%.

Active site architecture of an acetyl xylan esterase indicates a novel cold adaptation strategy.

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.

LncRNA PCED1B-AS1 Upregulation in Hepatocellular Carcinoma and Regulation of the miR-10a/BCL6 Axis to Promote Cell Proliferation.

Long noncoding RNA (lncRNA) PCED1B-AS1 has been characterized as an oncogene in glioma, but its role in hepatocellular carcinoma (HCC) is unknown. We explored the involvement of PCED1B-AS1 in HCC. Its expression in paired HCC and nontumor tissues from 62 HCC patients was determined by qRT-PCR. Correlations of PCED1B-AS1 with miR-10a and BCL6 were analyzed by Pearson's correlation coefficient. The patients were followed up for 5 years to analyze the prognostic value of PCED1B-AS1 for HCC. Interactions among PCED1B-AS1, miR-10a, and BCL6 were detected by overexpression experiments. Cell proliferation was analyzed by CCK-8 assay. We found the following. PCED1B-AS1 is upregulated in HCC, and its upregulation correlates with poor survival. Across HCC tissues, PCED1B-AS1 expression inversely correlates with miR-10a but positively correlates with BCL6. In HCC cells, overexpression of PCED1B-AS1 decreases miR-10a expression and increases BCL6 expression. Moreover, PCED1B-AS1 overexpression reduces the inhibitory effects of miR-10a overexpression on BCL6 expression and cell proliferation. PCED1B-AS1 is upregulated in HCC and regulates the miR-10a/BCL6 axis to promote cell proliferation.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects.

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent.IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Dual Pathways of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trafficking Modulate the Selective Exclusion of Uncleaved Oligomers from Virions.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail, and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: (i) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that cross-linked efficiently, and was recognized by broadly neutralizing antibodies; (ii) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and (iii) the larger population of uncleaved Env lacked complex carbohydrates, cross-linked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more "open" Env oligomers reached the cell surface in the presence of brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses toward conformationally flexible, uncleaved Env without compromising virus infectivity.IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus type 1 (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this nonfunctional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.

Combined strategy of knowledge-based rule selection and historical data percentile-based range determination to improve an autoverification system for clinical chemistry test results.

BACKGROUND: Current autoverification, which is only knowledge-based, has low efficiency. Regular historical data analysis may improve autoverification range determination. We attempted to enhance autoverification by selecting autoverification rules by knowledge and ranges from historical data. This new system was compared with the original knowledge-based system. METHODS: New types of rules, extreme values, and consistency checks were added and the autoverification workflow was rearranged to construct a framework. Criteria for creating rules for extreme value ranges, limit checks, consistency checks, and delta checks were determined by analyzing historical Zhongshan laboratory data. The new system's effectiveness was evaluated using pooled data from 20 centers. Efficiency improvement was assessed by a multicenter process. RESULTS: Effectiveness was evaluated by the true positive rate, true negative rate, and overall consistency rate, as compared to manual verification, which were 77.55%, 78.53%, and 78.3%, respectively for the new system. The original overall consistency rate was 56.2%. The new pass rates, indicating efficiency, were increased by 19%-51% among hospitals. Further customization using individualized data increased this rate. CONCLUSIONS: The improved system showed a comparable effectiveness and markedly increased efficiency. This transferable system could be further improved and popularized by utilizing historical data from each hospital.

Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic.

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.

Antigenicity and Immunogenicity of HIV-1 Envelope Trimers Complexed to a Small-Molecule Viral Entry Inhibitor.

Small-molecule viral entry inhibitors, such as BMS-626529 (BMS-529), allosterically block CD4 binding to HIV-1 envelope (Env) and inhibit CD4-induced structural changes in Env trimers. Here, we show that the binding of BMS-529 to clade C soluble chimeric gp140 SOSIP (ch.SOSIP) and membrane-bound trimers with intact transmembrane domain (gp150) prevented trimer conformational transitions and enhanced their immunogenicity. When complexed to BMS-529, ch.SOSIP trimers retained their binding to broadly neutralizing antibodies (bNAbs) and to their unmutated common ancestor (UCA) antibodies, while exposure of CD4-induced (CD4i) non-bNAb epitopes was inhibited. BMS-529-complexed gp150 trimers in detergent micelles, which were isolated from CHO cells, bound to bNAbs, including UCA and intermediates of the CD4 binding site (bs) CH103 bNAb lineage, and showed limited exposure of CD4i epitopes and a glycosylation pattern with a preponderance of high-mannose glycans. In rabbits, BMS-529-complexed V3 glycan-targeting ch.SOSIP immunogen induced in the majority of immunized animals higher neutralization titers against both autologous and select high mannose-bearing heterologous tier 2 pseudoviruses than those immunized with the noncomplexed ch.SOSIP. In rhesus macaques, BMS-529 complexed to CD4 bs-targeting ch.SOSIP immunogen induced stronger neutralization against tier 2 pseudoviruses bearing high-mannose glycans than noncomplexed ch.SOSIP trimer immunogen. When immunized with gp150 complexed to BMS-529, rhesus macaques showed neutralization against tier 2 pseudoviruses with targeted glycan deletion and high-mannose glycan enrichment. These results demonstrated that stabilization of Env trimer conformation with BMS-529 improved the immunogenicity of select chimeric SOSIP trimers and elicited tier 2 neutralizing antibodies of higher potency than noncomplexed trimers.IMPORTANCE Soluble forms of HIV-1 envelope trimers exhibit conformational heterogeneity and undergo CD4-induced (CD4i) exposure of epitopes of non-neutralizing antibodies that can potentially hinder induction of broad neutralizing antibody responses. These limitations have been mitigated through recent structure-guided approaches and include trimer-stabilizing mutations that resist trimer conformational transition and exposure of CD4i epitopes. The use of small-molecule viral inhibitors that allosterically block CD4 binding represents an alternative strategy for stabilizing Env trimer in the pre-CD4-triggered state of both soluble and membrane-bound trimers. In this study, we report that the viral entry inhibitor BMS-626529 restricts trimer conformational transition and improves the immunogenicity of select Env trimer immunogens.

Long-Acting BMS-378806 Analogues Stabilize the State-1 Conformation of the Human Immunodeficiency Virus Type 1 Envelope Glycoproteins.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)3] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.