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.
Highlights
The first reported human infected with the highly pathogenic avian influenza (HPAI)H5N1 virus was recorded in Hong Kong, 1997 [1]
Results indicated that dendritic cells (DCs)-SIGN expression in Raji cells significantly promoted H5N1-pseudo-type viruses (PVs) infection to the target Madin-Darby canine kidney (MDCK) cells (p < 0.01), while the promoting effect could be significantly blocked by pre-treatment of DC-SIGN mAbs on the capture cells (p < 0.05) (Figure 6B)
Similar significant and additive reducing effect was observed in H5N1-PVs carrying N27Q + N39Q or N27Q + N39Q + Nst1ra8i1nQw(aps f
Summary
The first reported human infected with the highly pathogenic avian influenza (HPAI)H5N1 virus was recorded in Hong Kong, 1997 [1]. The first reported human infected with the highly pathogenic avian influenza (HPAI). The spread of HPAI H5N1 viruses in poultry and sporadic human infections are perceived as a potential pandemic threat [2,3]. There are several clades and subclades being identified based on their genetic and antigenic variation of the viral haemagglutinin (HA5) and neuraminidase (NA1) genes. Clades 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7 have continued to evolve [4]. Clade 2.3.2 is widely distributed in Asian countries. Most H5N1 human infection results from direct contact with infected poultry [5]. Between 2003 and October 2020, there have been 861 human cases and 455 deaths reported by the World Health Organization (WHO) [6]
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