Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice.IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment.

COVID-19, Renin-Angiotensin System and Endothelial Dysfunction.

The newly emergent novel coronavirus disease 2019 (COVID-19) outbreak, which is caused by SARS-CoV-2 virus, has posed a serious threat to global public health and caused worldwide social and economic breakdown. Angiotensin-converting enzyme 2 (ACE2) is expressed in human vascular endothelium, respiratory epithelium, and other cell types, and is thought to be a primary mechanism of SARS-CoV-2 entry and infection. In physiological condition, ACE2 via its carboxypeptidase activity generates angiotensin fragments (Ang 1-9 and Ang 1-7), and plays an essential role in the renin-angiotensin system (RAS), which is a critical regulator of cardiovascular homeostasis. SARS-CoV-2 via its surface spike glycoprotein interacts with ACE2 and invades the host cells. Once inside the host cells, SARS-CoV-2 induces acute respiratory distress syndrome (ARDS), stimulates immune response (i.e., cytokine storm) and vascular damage. SARS-CoV-2 induced endothelial cell injury could exacerbate endothelial dysfunction, which is a hallmark of aging, hypertension, and obesity, leading to further complications. The pathophysiology of endothelial dysfunction and injury offers insights into COVID-19 associated mortality. Here we reviewed the molecular basis of SARS-CoV-2 infection, the roles of ACE2, RAS signaling, and a possible link between the pre-existing endothelial dysfunction and SARS-CoV-2 induced endothelial injury in COVID-19 associated mortality. We also surveyed the roles of cell adhesion molecules (CAMs), including CD209L/L-SIGN and CD209/DC-SIGN in SARS-CoV-2 infection and other related viruses. Understanding the molecular mechanisms of infection, the vascular damage caused by SARS-CoV-2 and pathways involved in the regulation of endothelial dysfunction could lead to new therapeutic strategies against COVID-19.

C-type Lectin CD209L/L-SIGN and CD209/DC-SIGN: Cell Adhesion Molecules Turned to Pathogen Recognition Receptors.

C-type lectin CD209/DC-SIGN and CD209L/L-SIGN proteins are distinct cell adhesion and pathogen recognition receptors that mediate cellular interactions and recognize a wide range of pathogens, including viruses such as SARS, SARS-CoV-2, bacteria, fungi and parasites. Pathogens exploit CD209 family proteins to promote infection and evade the immune recognition system. CD209L and CD209 are widely expressed in SARS-CoV-2 target organs and can contribute to infection and pathogenesis. CD209 family receptors are highly susceptible to alternative splicing and genomic polymorphism, which may influence virus tropism and transmission in vivo. The carbohydrate recognition domain (CRD) and the neck/repeat region represent the key features of CD209 family proteins that are also central to facilitating cellular ligand interactions and pathogen recognition. While the neck/repeat region is involved in oligomeric dimerization, the CRD recognizes the mannose-containing structures present on specific glycoproteins such as those found on the SARS-CoV-2 spike protein. Considering the role of CD209L and related proteins in diverse pathogen recognition, this review article discusses the recent advances in the cellular and biochemical characterization of CD209 and CD209L and their roles in viral uptake, which has important implications in understanding the host-pathogen interaction, the viral pathobiology and driving vaccine development of SARS-CoV-2.

NMR evidence for oligosaccharide release from the dendritic-cell specific intercellular adhesion molecule 3-grabbing non-integrin-related (CLEC4M) carbohydrate recognition domain at low pH.

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin-related (DC-SIGNR), also known as liver/lymph node-specific intercellular adhesion molecule 3-grabbing non-integrin, CLEC4M, CD209L, and CD299, is a Ca(2+) -dependent lectin that has been implicated in increasing the infection rates of several viruses, including HIV, but the physiological role of DC-SIGNR in healthy cells is currently not known with certainty. A close homologue of DC-SIGNR, dendritic-cell specific intercellular adhesion molecule 3-grabbing non-integrin, has been shown to act as a recycling endocytic receptor, which binds pathogens at the cell's surface and then releases them in the low pH environment of endosomal compartments. However, it is currently under debate in the literature as to whether DC-SIGNR plays a similar role. In this work, we used NMR to explore whether the DC-SIGNR carbohydrate recognition domain (CRD) shows any pH dependence in its ability to bind carbohydrates and Ca(2+) . We found clear evidence of reduced or abolished CRD-binding affinities for three different glycans at low pH (4.2) as compared to neutral pH (6.8). We also report the assignment of the DC-SIGNR CRD in the apo form, and use these new results to characterize the degree of structural rearrangement upon binding (or release) of Ca(2+) . Finally, we report a differential effect of pH on the affinities of glycans containing mannose exclusively versus glycans containing GlcNAc moieties. Our results lead us to propose that the DC-SIGNR CRD rapidly and reversibly releases glycan ligands and Ca(2+) at reduced pH (behaviour that would be expected for an endocytic receptor), and that the binding of mannose-containing oligosaccharides is more strongly affected by pH than the binding of GlcNAc-containing oligosaccharides.