(a) Representative immunofluorescence images from three independent experiments of ANDV-, HTNV-, SEOV-, PUUV-, PHV- and TULV-infected HUVEC after exposure to staurosporine (2?M) for approximately 4?hours

(a) Representative immunofluorescence images from three independent experiments of ANDV-, HTNV-, SEOV-, PUUV-, PHV- and TULV-infected HUVEC after exposure to staurosporine (2?M) for approximately 4?hours. for caspase-3 and granzyme B. Recombinant N protein from ANDV, PUUV R-121919 and the HFRS-causing Dobrava virus strongly inhibited granzyme B activity and also, to certain extent, caspase-3 activity. Taken together, this study demonstrates that six different orthohantaviruses inhibit apoptosis, suggesting this to be a general feature of orthohantaviruses likely serving as a mechanism of viral immune evasion. Introduction Orthohantaviruses, of the order and previously known as hantaviruses, are small single-stranded negative-sense RNA viruses with a tri-segmented genome (S, M and L segments) encoding four to five proteins. The S segment encodes a nucleocapsid protein (N), the M segment two R-121919 glycoproteins (Gn and Gc), and the L segment an RNA dependent RNA polymerase1C5. Additionally, the S segment of some orthohantaviruses also encodes a non-structural protein called NSs5. The natural hosts for orthohantaviruses are various small animals, mainly rodents, but also moles, shrews and bats, and as recently shown fishes and reptiles too1C6. Each distinct orthohantavirus primarily infects one specific animal species7,8. Orthohantaviruses establish life-long infection in their respective natural hosts2,5. However these viruses cause strong immune responses in the natural host9 and it is currently not well known how orthohantaviruses avoid being eradicated. Orthohantaviruses have a worldwide distribution4,5. At present more Nr2f1 than fifty different orthohantaviruses, whereof twenty are pathogenic to humans, have been identified2. Rodent-borne orthohantaviruses can cause hantavirus pulmonary syndrome (HPS; also known as hantavirus cardiopulmonary syndrome (HCPS)) and hemorrhagic fever with renal syndrome (HFRS)1C3. Three different rodent subfamilies C and C harbor the majority of the known orthohantaviruses, including all known HPS- and HFRS-causing viruses as well as several non-pathogenic ones. Phylogenetic analyses have shown that rodents cluster with certain mole- (do not cause any direct cytopathic effects23,24 and despite the robust immune activation observed in patients, infected endothelial cells remain undamaged25C27. Apoptosis is a well-regulated mechanism to eliminate cells, including virus-infected cells or tumorigenic cells. Apoptosis plays an important role in restricting the dissemination of pathogens, such as viruses, throughout the body. Caspases (cysteine-dependent aspartate-directed proteases) act as main orchestrators of apoptosis. These proteases are present as inactive zymogens requiring cleavage and subsequent oligomerization to become active. During apoptosis, caspase-3 is activated and cleaves several cellular key R-121919 protein components, such as the poly ADP-ribose polymerase (PARP)28,29. Caspase-3 is necessary for chromatin condensation and DNA fragmentation, two typical hallmarks of apoptosis30. Because of the crucial role played in determining cell fate, the action of caspases is regulated at multiple levels, both prior to and after activation31C33. Given the importance of apoptosis-inducing pathways in cellular anti-viral defense, it is not surprising that some viruses have been R-121919 shown to interfere with one or more components of these pathways33C40. Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T cells (CTL), represent important components of the immune response towards virus infections. Both cell types kill virus-infected cells in a similar manner, mainly via cytotoxic granule-mediated activation of R-121919 target cell apoptosis. The cytotoxic granules contain granzymes, which upon release into target cells cleave certain cellular substrates thereby activating cell death pathways41. Mainly, this occurs via direct granzyme B activation of caspase-342,43. Granzyme B has also been reported to induce programmed cell death in a caspase-independent manner41,44. Orthohantavirus-infected patients show robust cytotoxic lymphocyte responses encompassing a long-lived NK cell response including specific expansion of NKG2C+ NK cells45 and strong virus-specific cytotoxic CD8+ T cell responses at onset.