Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

Abstract

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004–2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.

Heat Shock Protein Member 8 (HSPA8) Is Involved in Porcine Reproductive and Respiratory Syndrome Virus Attachment and Internalization

“…ELISA was performed to evaluate the direct interaction between HSPA8 and PRRSV virions according to a previous study with minor modifications ( 60 ). Briefly, the 96-well ELISA plates (Corning, New York, USA) were coated with the purified recombinant GST-tagged proteins (0.1, 0.2, 0.4, 0.8 μM/well) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 4°C for 12 h, and then blocked with 5% bovine serum albumin (BSA) at 37°C for 1 h. After three washes, the plates were incubated with PRRSV virions (10 8.1 TCID 50 /mL in PBS, 100 μL/well) at room temperature (RT) for 2 h. To detect the interaction, anti-GP5 MAb at 1:3,000 was added to each well and incubated at 37°C for 1 h. The wells were washed three times and incubated with HRP-conjugated goat anti-mouse IgG antibody at 1:10,000 at 37°C for 1 h. The reactions were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) and terminated with H 2 SO 4 .…”

DC-SIGN Receptor Level in Rheumatoid Arthritis Patients in Baghdad; Serological study

“…Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209) is a type II CLRs that acts as an adhesion molecule situated on dendritic cells (DCs), which recognizes antigens and can promote TLR to induced activation of NF-κB 6 . DC-SIGN is participating in DCs migration and activation of T lymphocytes, in addition, it can be a goal of some microorganisms or tumor cells, which may result in avoidance from immune surveillance or immune suppression 7 . Serum levels of DC-SIGN in RA are not yet determined in the Iraqi patients, therefore the purpose of our study is assess the serum levels of DC-SIGN in a sample of Iraqi RA patients, which might have a broad influence on host immune responses.…”

Targeting SARS-CoV-2-Platelet Interactions in COVID-19 and Vaccine-Related Thrombosis

“…There are a number of C-type lectins found on platelets including DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) and CLEC (C-type lectin-like receptor) 2. DC-SIGN, which binds HIV ( Chaipan et al, 2006 ), DENV ( Tomo et al, 2018 ), Ebola virus ( Alvarez et al, 2002 ), Hepatitis virus ( Pöhlmann et al, 2003 ) and influenza H1N1 ( Londrigan et al, 2011 ) and H5N1 ( Yang et al, 2021 ), is implicated in platelet activation. CLEC-2 and 5A are also important in binding to viruses ( Sung and Hsieh, 2019 ) such as HIV ( Chaipan et al, 2006 ).…”