The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage).cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys.trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin⁻Sca-1⁺c-Kit⁺ (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.

DC-SIGN is a C-type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV-1, HIV-2, and SIV. Inaddition, the ability of DC-SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T-cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC-SIGN, weperformed a detailed analysis of DC-SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC-SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC-SIGNexpression in adult and fetal tissues except that DC-SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC-SIGN was observed primarily on immature (CD83-negative)DCs. Secondly, in the peripheral blood, we found expression of DC-SIGNon a small subset of BDCA-2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC-SIGN-positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2). Triple-label confocal microscopy indicated that DC-SIGN was colocalizedwith BDCA-2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC-SIGN can be up-regulated onmonocyte-derived macrophages upon exposure to the Th2 cytokine, IL-13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC-SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC-SIGN expression maybe involved in the Th2 axis of immunity.

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans , while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans . However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.