The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Abstract

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

“…In brief, 293T cells transfected with expression vectors for MERS-CoV S, VSV-G (positive control) or empty expression vector (negative control) were inoculated with VSV*ΔG-fLuc for 1 h before being washed with PBS and further incubated for 16 h with culture medium that was supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) (except for cells expressing VSV-G). The produced VSVpp were inoculated onto BHK-21 cells expressing WT or mutant DPP4, or no DPP4 (empty expression vector, negative control) and incubated for 16-18 h before fLuc activity in cell lysates was quantified as an indicator for transduction efficiency using the Beetle-Juice kit (PJK) and a plate luminometer (Hidex) [41].…”

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism

“…Exposure of recombinant HL17 and HL18 to low pH did not render the proteins sensitive to degradation by trypsin, in contrast to conventional HA subtypes (11). However, infection of bat cells with HL-pseudotyped VSV occurred in a pH-dependent manner (12,13). Using the same approach, it was also shown that proteolytic activation of the viral glycoproteins is essential to obtain infectious pseudotyped viruses (12,13).…”

“…However, infection of bat cells with HL-pseudotyped VSV occurred in a pH-dependent manner (12,13). Using the same approach, it was also shown that proteolytic activation of the viral glycoproteins is essential to obtain infectious pseudotyped viruses (12,13). Moreover, HL17-and HL18-pseudotyped viruses revealed a restricted cell tropism, because only certain bat cell lines were found to be susceptible to infection.…”

“…Moreover, HL17-and HL18-pseudotyped viruses revealed a restricted cell tropism, because only certain bat cell lines were found to be susceptible to infection. In one study, Madin-Darby canine kidney (MDCK) cells were successfully infected with HL-pseudotyped VSV (12); however, this finding was not confirmed by others (13). Susceptible human cells could not be identified yet.…”

“…The putative receptor-binding pockets of HL17 and HL18 contain several acidic amino acid residues, rendering the binding to negatively charged molecules such as sialic acid unlikely (8,11). Accordingly, infection of bat cells with HL-pseudotyped vesicular stomatitis virus (VSV) was not affected if the cells were pretreated with sialidase (12,13). In an attempt to identify the putative receptor for HL17, a chip covering more than 600 different glycans was screened with recombinant soluble HL17 protein, but no binding to carbohydrates was observed (14).…”