SARS CoV-2 Variants and Spike Mutations Involved in Second Wave of COVID-19 Pandemic in India

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole genome sequences of SARS CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24, 260 mutations in the S protein, based on which 265 pango lineages could be categorised. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, Jammu & Kashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of >1%; 70, 18, 7, 3, 9, 4, and 1 (N=112) such mutations were observed in the N -terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2, and cytoplasmic domains of the S protein. Interestingly, 13.39% (N=15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2%(N=3) were known to be vaccine breakthrough mutations; and 0.89%(N=1) were known to escape neutralising antibodies. Biological significance of 82% (N=92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

Genetic Polymorphisms of the TGFB1 Signal Peptide and Promoter Region: Role in Wilms Tumor Susceptibility?

The aim of the present study was to investigate the rs1800468 (G-800A), rs1800469 (C-509T), rs1800470 (C29T), and rs1800471 (G74C) TGFB1 genetic polymorphisms and their haplotype structures in patients with Wilms Tumor (WT) and neoplasia-free controls. The genomic DNA was extracted from 35 WT patients and 160 neoplasia-free children, and the TGFB1 polymorphisms were genotyped by polymerase chain reaction, followed by restriction fragment length polymorphism. The haplotype structures were inferred, and permutation and logistic regression tests were performed to check for differences in haplotype distribution between the control and WT individuals. Positive associations were found in the recessive model for rs1800469 T allele (OR: 8.417; 95% CI: 3.177 to 22.297; P < 0.001) and for the rs1800470 C allele (OR: 3.000; 95% CI: 1.296 to 6.944; P = 0.01). Haplotype analysis revealed a significant negative association between GCTG and WT (OR: 0.236, 95% CI: 0.105 to 0.534; P = 0.0002); by contrast, the GTTG haplotype was associated with increased risk for WT (OR: 12.0; 95% CI: 4.202 to 34.270; P < 0.001). Furthermore, rs1800469 was negatively correlated with tumor size and a trend toward a positive correlation for capsular invasion was observed in the dominant model (Tau-b: −0.43, P = 0.02 and tau-b: 0.5, P = 0.06, respectively). This is the first study with rs1800468, rs1800469, rs1800470, and rs1800471 TGFB1 polymorphisms in WT, and our results suggest that the TGFB1 promoter and signal peptide region polymorphisms may be associated with WT susceptibility and clinical presentation.

Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated −5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6−8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo­ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

The ER chaperones BiP and Grp94 regulate the formation of insulin-like growth factor 2 (IGF2) oligomers

While cytosolic Hsp70 and Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp70 and Hsp90 paralogs (BiP and Grp94) recognize clients and influence their folding. Here, we examine how BiP and Grp94 influence the folding of insulin-like growth factor 2 (IGF2). Full-length proIGF2 is composed of an insulin-like hormone and an E-peptide that has sequence characteristics of an intrinsically disordered region. We find that the E-peptide region allows proIGF2 to form oligomers. BiP and Grp94 influence both the folding and the oligomerization of proIGF2. BiP and Grp94 exert a similar holdase function on proIGF2 folding by preferentially binding the proIGF2 unfolded state, rather than stabilizing specific folding intermediates and changing the proIGF2 folding process. In contrast, BiP and Grp94 exert counteracting effects on proIGF2 oligomerization. BiP suppresses proIGF2 oligomerization under both ADP and ATP conditions. Interestingly, Grp94 can enhance proIGF2 oligomerization when Grp94 adopts an open conformation (ADP conditions), but not when Grp94 is in the closed conformation (ATP conditions). We propose that BiP and Grp94 regulate the assembly of proIGF2 oligomers, and that regulated oligomerization may enable proIGF2 to be effectively packaged for export from the ER to the Golgi.

In Silico Structure-Based Repositioning of Approved Drugs for Spike Glycoprotein S2 Domain Fusion Peptide of SARS-CoV-2: Rationale from Molecular Dynamics and Binding Free Energy Calculations

The present study provides the structural identification of the viable binding residues of the SARS-CoV-2 S2 fusion peptide region, which holds prime importance in the virus’s host cell fusion and entry mechanism. The classical molecular mechanics simulations were set on values that mimic physiological standards for a good approximation of the dynamic behavior of selected drugs in biological systems. The drug molecules screened and analyzed here have relevant antiviral properties, which are reported here and which might hint toward their utilization in the coronavirus disease 2019 (COVID-19) pandemic owing to their attributes of binding to the fusion protein binding region shown in this study.

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis

ABSTRACT HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens. IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

In silico Vaccine Design against Dengue Virus Type 2 Envelope Glycoprotein

Dengue fever is caused by the mosquito-borne virus termed (DENV). However, DENV-2 has been identified as the most prevalent amongst the Indonesian pediatric urban population, in contrast with the other four serotypes. Therefore, it is important to reduce severe infection risk by adopting preventive measures, including through vaccine development. The aim of this study, therefore is to use various in silico tools in the design of epitope-based peptide vaccines (T-cell and B-cell types), based on the DENV-2 envelope glycoprotein sequences available. Therefore, in silico methods were adopted in the analysis of the retrieved protein sequences. This technique was required to determine the most immunogenic protein, and is achieved through conservancy analysis, epitope identification, molecular simulation, and allergenicity assessment. Furthermore, B4XPM1, and KAWLVHRQW were identified from positions 204-212, while the 77 to 85 peptide region was considered the most potent T-cell and B-cell epitopes. The interaction between KAWLVHRQW and HLA-C*12:03 occurs with maximum population coverage, alongside high conservancy (96.98%) and binding affinity. These results indicated a potential for the designed epitopes to demonstrate high immunity against DENV-2.