scholarly journals Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

2021 ◽  
Vol 17 (10) ◽  
pp. e1009996
Author(s):  
André Volland ◽  
Michael Lohmüller ◽  
Emmanuel Heilmann ◽  
Janine Kimpel ◽  
Sebastian Herzog ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Single Point ◽  
Lymphocytic Choriomeningitis ◽  
Single Point Mutation ◽  
Biosynthesis Pathway ◽  
Virus Binding ◽  
Virus Attachment ◽  
A Genome ◽  
Key Factor ◽  
Lcmv Infection

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

Multilevel Evolution: The Fate of Duplicated Genes

2002 ◽  
Vol 216 (1) ◽  
Author(s):  
Paulien Hogeweg
Keyword(s):  
Information Integration ◽  
Large Scale ◽  
Evolutionary Dynamics ◽  
Single Point ◽  
Gene Content ◽  
Minor Role ◽  
Single Point Mutation ◽  
Duplicated Genes ◽  
A Genome ◽  
Multilevel Evolution

Biological evolution is a multilevel process and should be studied as such. A first, important step in studying evolution in this way has been the work of Peter Schuster and co-workers on RNA evolution. For RNA the genotype-phenotype mapping can be calculated explicitly. The resulting evolutionary dynamics is dominated by neutral paths, and the potential of major change by a single point mutation.Examining whole genomes, of which about 60 are now available, we see that gene content of genomes is changing relatively rapidly: gene duplication, gene loss and gene generation is ubiquitous. In fact, it seems that point-mutations play a relatively minor role, relative to changes in gene regulation and gene content in adaptive evolution.Large scale micro-array studies, in which the expression of every gene can be measured simultaneously, give a first glimpse of the `division of labor´ between duplicated genes. A preliminary analysis suggests that differential expression is often the primary event which allows duplicated genes to be maintained in a genome, but alternate routes also exist, most notably on the one hand the mere need of a lot of product, and on the other hand differentiation within multi-protein complexes consisting of homologous genes.I will discuss these results in terms of multilevel evolution. in particular in terms of information integration and the alternatives of `individual based´

scholarly journals Characterization and engineering of S100A12–heparan sulfate interactions

Glycobiology ◽  
2020 ◽  
Vol 30 (7) ◽  
pp. 463-473
Author(s):  
Xiaoxiao Zhang ◽  
Chihyean Ong ◽  
Guowei Su ◽  
Jian Liu ◽  
Ding Xu
Keyword(s):  
Heparan Sulfate ◽  
Binding Site ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Single Point ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Unexpected Finding

Abstract S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12–HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12–HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single-point mutation. Using a HS oligosaccharide microarray, we demonstrated that the N43K mutant displayed markedly enhanced selectivity toward longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

scholarly journals The Fusion Glycoprotein of Human Respiratory Syncytial Virus Facilitates Virus Attachment and Infectivity via an Interaction with Cellular Heparan Sulfate

2000 ◽  
Vol 74 (14) ◽  
pp. 6442-6447 ◽  
Author(s):  
Steven A. Feldman ◽  
Susette Audet ◽  
Judy A. Beeler
Keyword(s):  
Respiratory Syncytial Virus ◽  
Heparan Sulfate ◽  
Specific Activity ◽  
Recombinant Vaccinia Virus ◽  
Enzymatic Digestion ◽  
Human Respiratory Syncytial Virus ◽  
Virus Binding ◽  
Virus Attachment ◽  
Fusion Glycoprotein ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED50] = 0.28 ± 0.11 μg/ml) andcp-52 (ED50 = 0.55 ± 0.14 μg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.

scholarly journals Widespread Mutations in Voltage-Gated Sodium Channel Gene of Cimex lectularius (Hemiptera: Cimicidae) Populations in Paris

2021 ◽  
Vol 18 (2) ◽  
pp. 407
Author(s):  
Mohammad Akhoundi ◽  
Dahlia Chebbah ◽  
Denis Sereno ◽  
Anthony Marteau ◽  
Julie Jan ◽  
...  
Keyword(s):  
Single Point ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Homozygous Mutation ◽  
Knockdown Resistance ◽  
Bed Bugs ◽  
Bed Bug ◽  
Channel Gene ◽  
Blood Sucking ◽  
The Status

Bed bugs, Cimex lectularius and C. hemipterus, are common blood-sucking ectoparasites of humans with a large geographical distribution, worldwide. In France, little is known about the status of bed bugs’ infestation and their resistance to insecticides, particularly, pyrethroids. Here, we aimed to find mutations in the kdr gene, known to be involved in resistance to insecticides. We gathered bed bugs from various infested locations, including 17 private houses, 12 HLM building complex, 29 apartments, 2 EHPAD, and 2 immigrants’ residences. A total of 1211 bed bugs were collected and morphologically identified as C. lectularius. Two fragments of the kdr gene, encompassing codons V419L and L925I, were successfully amplified for 156 specimens. We recorded sense mutation in the first amplified fragment (kdr1) in 89 out of 156 (57%) samples, in which in 61 out of 89 (68.5%) sequences, a change of valine (V) into leucine (L) V419L was observed. Within the second fragment (kdr2), a homozygous mutation was recorded in 73 out of 156 (46.7%) specimens at the codon 925. At this position, 43 out of 73 (58.9%) specimens had a sense mutation leading to the replacement of leucine (L) by isoleucine (I). Among 162 mutant sequences analyzed (89 for the kdr1 fragment and 73 for the kdr2 one), we detected single point mutation in 26.6%, while 73.4% presented the mutation in both kdr1 and kdr2 fragments. All modifications recorded in bed bug populations of Paris are described to be involved in the knockdown resistance (kdr) against pyrethroids.

scholarly journals A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

2021 ◽  
Vol 7 (7) ◽  
pp. 553
Author(s):  
Bin Gao ◽  
Shunyi Zhu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Single Point ◽  
Binding Domain ◽  
Host Cells ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Microsporum Canis ◽  
Receptor Binding Domain ◽  
Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

scholarly journals Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bingran Wang ◽  
Tiancheng Lou ◽  
Lingling Wei ◽  
Wenchan Chen ◽  
Longbing Huang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Single Point ◽  
Sodium Dodecyl ◽  
Cross Resistance ◽  
Molecular Characteristics ◽  
Single Point Mutation ◽  
Wide Range ◽  
Significant Difference ◽  
Leaf Blights ◽  
High Level

AbstractAlternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

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