Research Article: Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

Date Published: February 23, 2015

Publisher: Public Library of Science

Author(s): Farah El Najjar, Levi Lampe, Michelle L. Baker, Lin-Fa Wang, Rebecca Ellis Dutch, Shan-Lu Liu.


Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

Partial Text

In the past twenty years, bats of different species have been recognized as important hosts of viruses from different families including rhabdoviruses [1–3], coronaviruses [4–9], filoviruses [10–12], flaviviruses [13,14], orthomyxoviruses [15–17], paramyxoviruses [18,19] and others [20,21]. Numerous studies have shown that bats not only harbor a large number of viruses, but are also a major source for the emergence and transmission of viruses that cause highly pathogenic infectious diseases in humans, most importantly Severe Acute Respiratory Syndrome-like coronavirus (SARS-like CoV) [7], Ebola virus [10,22] and the henipaviruses, Hendra virus [23–26] and Nipah virus [27–29], which are members of the paramyxovirus family. Hendra virus first emerged in 1994 in Australia in an outbreak that occurred in horses [30], and more than thirty subsequent outbreaks have occurred, with a total of four human deaths associated with the virus infection [31,32]. Another closely related virus, Nipah virus was identified in Malaysia in 1999 causing an outbreak of viral encephalitis [33]; with additional outbreaks showing high mortality rates that reached 70%. Several species of bats within the genus Pteropus, commonly known as flying foxes, have been confirmed as the natural primary reservoir of henipaviruses [23,25,27,34–36]. Cedar virus, a novel henipavirus that does not seem to cause clinical disease in several animals which are known to be susceptible to Hendra and Nipah viruses, was identified recently and also has Pteropus bats as its natural reservoir [37]. Recent evidence suggests that henipaviruses are also present in non-Pteropus fruit bats in Africa [38,39]. Despite the important role of bats in the emergence of henipaviruses and other highly pathogenic viruses, very little is known about the viral life cycle or virus-host interactions in this natural reservoir.

Bats have recently been shown to carry a number of novel viruses [94]; however, our knowledge of the natural history of viruses in their bat reservoir host and the special features of bats that allow them to co-exist with this wide range of viruses is limited. Bats and bat-derived cells are susceptible to infection by many viruses including filoviruses, paramyxoviruses, coronaviruses and influenza virus [74,95] indicating that bats have the necessary cellular factors to mediate many viral infections. Cellular proteases play an essential role in proteolytic activation of the majority of viral glycoproteins and in the spread of infection, but very little is currently known about the protease profile of the bat hosts. Interestingly, a number of bat-borne viruses utilize the endosomal cathepsin proteases during their life cycle [47,48,63–65], in contrast to the more common use of furin proteases for intracellular viral glycoprotein processing. To address the ability of bat cells to proteolytically process viral fusion proteins, we examined the proteolytic processing of the PIV5 F protein, normally cleaved by furin, and the Hendra virus F protein, normally cleaved by cathepsin L, in cells derived from two species of bats of the Pteropodidae family. We showed that P. alecto and R. aegyptiacus have homologues of cathepsin and furin proteases capable of cleaving and activating cathepsin-dependent (Hendra virus F) and furin-dependent (PIV5 F) viral fusion proteins. This finding is consistent with previous studies showing that cells from different bat species can cleave glycoproteins of some viruses such as Ebola virus [96], and an African henipavirus [97,98]. Our data also indicate that the requirements for proteolytic processing of Hendra virus F in bat cells are analogous to those previously determined in Vero cells [47,52]. Temperature reduction experiments or inhibition of cathepsin L prevented both cleavage of Hendra virus F and syncytia formation (data not shown), indicating that vesicular trafficking and a bat homolog of cathepsin L are involved in activation of Hendra virus F in bat cells. In addition, we did not detect a significant difference in the kinetics of Hendra F cleavage in PaKi or R06E compared to Vero cells and levels of cleaved F1 on the cell surface of Vero cells and bat cells were similar (data not shown). These results indicate that Hendra virus F trafficking in bat cells is analogous to that in Vero cells, suggesting that Hendra virus evolved its dependence on cathepsin L to mediate infection through adaptation in its bat natural host.