Christina B. Karsten

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Abstract Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation.

Recruitment of the tumour suppressor protein p73 by Kaposi’s Sarcoma Herpesvirus latent nuclear antigen contributes to the survival of primary effusion lymphoma cells

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman’s disease (MCD). The KSHV latency-associated nuclear antigen-1 (LANA), required for the replication and maintenance of latent viral episomal DNA, is involved in the transcriptional regulation of viral and cellular genes and interacts with different cellular proteins, including the tumour suppressor p53. Here, we report that LANA also recruits the p53-related nuclear transcription factor p73, which influences cellular processes like DNA damage response, cell cycle progression and apoptosis. Both the full-length isoform TAp73α, as well as its dominant negative regulator ΔNp73α, interact with LANA. LANA affects TAp73α stability and sub-nuclear localisation, as well as TAp73α-mediated transcriptional activation of target genes. We observed that the small-molecule inhibitor Nutlin-3, which disrupts the interaction of p53 and p73 with MDM2, induces apoptotic cell death in p53 wild-type, as well as p53-mutant PEL cell lines, suggesting a possible involvement of p73. The small-molecule RETRA, which activates p73 in the context of mutant p53, leads to the induction of apoptosis in p53-mutant PEL cell lines. RNAi-mediated knockdown of p73 confirmed that these effects depend on the presence of the p73 protein. Furthermore, both Nutlin-3 and RETRA disrupt the LANA–p73 interaction in different PEL cell lines. These results suggest that LANA modulates p73 function and that the LANA–p73 interaction may represent a therapeutic target to interfere with the survival of latently KSHV-infected cells.

Influenza A virus does not encode a tetherin antagonist with Vpu-like activity and induces IFN-dependent tetherin expression in infected cells.

The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity.

Analysis of Ebola Virus Entry Into Macrophages

Abstract Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)–driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.

Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity

Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. The spike protein of MERS-CoV (MERS-S) facilitates viral entry into host cells, which depends on activation of MERS-S by cellular proteases. Proteolytic activation of MERS-S during viral uptake into target cells has been demonstrated. However, it is unclear whether MERS-S is also cleaved during S protein synthesis in infected cells and whether cleavage is required for MERS-CoV infectivity. Here, we show that MERS-S is processed by proprotein convertases in MERS-S–transfected and MERS-CoV–infected cells and that several RXXR motifs located at the border between the surface and transmembrane subunit of MERS-S are required for efficient proteolysis. However, blockade of proprotein convertases did not impact MERS-S–dependent transduction of target cells expressing high amounts of the viral receptor, DPP4, and did not modulate MERS-CoV infectivity. These results show that MERS-S is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for S protein activation. Efforts to inhibit MERS-CoV infection by targeting host cell proteases should therefore focus on enzymes that process MERS-S during viral uptake into target cells.

Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

Abstract Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein–pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo.

The role of the alternative coreceptor GPR15 in SIV tropism for human cells

Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4(+), CD8(+) and CD19(+) peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15(+) PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4(+), GPR15(+) cells is not responsible for the well documented ability of SIV to infect CCR5(-) blood cells.

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

ABSTRACT Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.

Exploiting glycan topography for computational design of Env glycoprotein antigenicity

Mounting evidence suggests that glycans, rather than merely serving as a “shield”, contribute critically to antigenicity of the HIV envelope (Env) glycoprotein, representing critical antigenic determinants for many broadly neutralizing antibodies (bNAbs). While many studies have focused on defining the role of individual glycans or groups of proximal glycans in bNAb binding, little is known about the effects of changes in the overall glycan landscape in modulating antibody access and Env antigenicity. Here we developed a systems glycobiology approach to reverse engineer the complexity of HIV glycan heterogeneity to guide antigenicity-based de novo glycoprotein design. bNAb binding was assessed against a panel of 94 recombinant gp120 monomers exhibiting defined glycan site occupancies. Using a Bayesian machine learning algorithm, bNAb-specific glycan footprints were identified and used to design antigens that selectively alter bNAb antigenicity as a proof-of concept. Our approach provides a new design strategy to predictively modulate antigenicity via the alteration of glycan topography, thereby focusing the humoral immune response on sites of viral vulnerability for HIV.