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

Abstract

Hantaviruses are a large group of RNA viruses that include known epidemic threats and other agents poised for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures alike 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. Previous work has implicated several cell-surface molecules, most notably β3- and β1-containing integrin heterodimers, decay-accelerating factor (DAF), and the cadherin superfamily protein protocadherin-1 (PCDH1), in hantavirus entry in endothelial cells, the major targets of viral infection in humans. Despite the fact that β3/β1 integrins have been presumed to be the major hantavirus entry receptors for over two decades, rigorous genetic evidence supporting their requirement, and that of DAF as an entry cofactor, is lacking. Here, we used CRISPR/Cas9 engineering to knock out four candidate hantaviral receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells. PCDH1 loss substantially reduced entry and infection by a subset of hantaviruses endemic to the Americas. In contrast, the loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by any of a large panel of hantaviruses tested. We conclude that the 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

“…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. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV, the primary etiologic agents of HCPS in the Americas, and is likely to play such a role for other New World hantaviruses shown to utilize PCDH1 for entry (6,32).…”