Mild reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant: First case report from Indonesia.

Background: Reinfection with SARS-CoV-2 has been well documented, yet little is known about the degree of protection a previous infection provides against reinfection, especially against Variants of Concern (VOC). Case presentation: Here we describe a case of an unvaccinated 49-year-old man who experienced two sequential SARS-CoV-2 infections with two different variants, as evidenced by genomic sequencing. The first episode was caused by the Pango lineage B.1.466.2 and resulted in severe COVID-19 with 5 days in an intensive care unit (ICU). The second episode occurred approximately 6 months later, during the Delta surge in Indonesia. Genomic analysis showed that the second infection was caused by the Delta variant (Pango lineage B.1.617.2) and resulted in mild disease that did not require hospitalization. No SARS-CoV-2 nucleic acid was detected between the two episodes, but both binding and neutralizing antibodies to SARS-CoV-2 were detected prior to the reinfection, with the second infection leading to an increase in the levels of antibody. Conclusion: We confirmed that the patient experienced a reinfection instead of persistent viral shedding from the first infection based on epidemiological, clinical, serological, and genomic analyses. Our case supports the hypothesis that SARS-CoV-2 reinfection may occur once antibody titers decrease or following the emergence of a new variant. The milder presentation in the patient's second infection deserves further investigation to provide a clear picture of the role of post-infection immunity in altering the course of subsequent disease.

The active and the inactive form of the hAT1 receptor have an identical ligand-binding environment: an MPA study on a constitutively active angiotensin II receptor mutant.

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT1)1 receptor mutant N111G-hAT1 which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT1 receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT1 series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.

Methionine proximity assay, a novel method for exploring peptide ligand-receptor interaction.

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor.

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.