Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

Abstract

Hantaviruses are RNA viruses with known epidemic threat and potential for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Despite the long-standing presumption that β3/β1-containing integrins are the major hantavirus entry receptors, rigorous genetic loss-of-function evidence supporting their requirement, and that of decay-accelerating factor (DAF), is lacking. Here, we used CRISPR/Cas9 engineering to knockout candidate hantavirus receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells, the major targets of viral infection in humans. The loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by a large panel of hantaviruses. By contrast, loss of protocadherin-1, a recently identified entry receptor for some hantaviruses, substantially reduced hantavirus entry and infection. We conclude that major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

Two point mutations in protocadherin-1 disrupt Andes hantavirus recognition and afford protection against lethal infection

“…Thus, F83L reduces PCDH1 binding to both ANDV and SNV Gn/Gc, but abolishes PCDH1 binding only to SNV Gn/Gc, concordant with its selective effect on entry and infection (Figure 1F). As shown previously, PCDH1 is dispensable for viral infection mediated by Old World hantavirus Gn/Gc (e.g., HTNV) (Figure 1F) (6,7). These findings, together with the conservation of F83 in PCDH1 orthologs from species known to be susceptible to New World hantaviruses (Figure 1C), provide evidence that Gn/Gc:PCDH1 recognition can influence the cellular host range of hantaviruses.…”

“…Virus-receptor interactions can influence viral entry, cell and tissue tropism, viral host range and pathogenesis, and afford attractive targets for antiviral countermeasures (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). We previously demonstrated that PCDH1 is an essential entry host factor for New World hantaviruses (6,7).…”

“…Virus-receptor interactions can influence viral entry, cell and tissue tropism, viral host range and pathogenesis, and afford attractive targets for antiviral countermeasures (22-31). We previously demonstrated that PCDH1 is an essential entry host factor for New World hantaviruses (6,7). Herein, we identified residues in the EC1 domain of PCDH1 critical for ANDV and SNV Gn/Gc engagement, including one that influences cellular host range, and demonstrated that Gn/Gc:PCDH1 recognition, not just PCDH1 per se , is required for development of lethal ANDV infection in Syrian hamsters.…”

“…‘Old World’ hantaviruses found primarily in Asia and Europe, such as Hantaan virus (HTNV) and Seoul virus (SEOV), can cause HFRS in humans (2). We recently identified protocadherin-1 (PCDH1), a member of the non-clustered protocadherins in the cadherin superfamily (4,5), as a clade-specific entry host factor for New World hantaviruses, including ANDV and SNV, that directly engages the viral Gn/Gc glycoprotein complex (6,7), which mediates cell entry (8). PCDH1 is expressed in the airway epithelium, where it colocalizes with E-cadherin at apical cell-cell contact sites (9-11), and in vascular endothelial cells, the primary targets for hantavirus infection (12).…”

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses

“…Recombinant vesicular stomatitis viruses rVSVs expressing enhanced green fluorescent protein (eGFP) and bearing Gn/Gc from HTNV, SEOV, DOBV, ANDV, SNV, CHOV, and VSV G were engineered, rescued, and propagated following published protocols (30,31,(47)(48)(49). rVSV carrying a phosphoprotein P fused to mNG and bearing PUUV Gn/Gc has been described previously (29).…”