Biology subjects

Schlagowski, S.

Publications and source records attributed to Schlagowski, S..

7 recordsLinked to original sources

bioRxiv2024

Glycoprotein K8.1 is Critical for Liver and Bone Marrow Tropism of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) in the Marmoset Infection Model

Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus with significant disease burden, particularly in Sub-Saharan Africa, where Kaposis sarcoma is prevalent. KSHV disproportionately affects immunocompromised individuals and men who have sex with men. A suitable animal model is critical for the development of a preventive vaccine, as it must replicate viral spread and immune interactions. The common marmoset (Callithrix jacchus) had previously been established as conducive to KSHV infection with non-BAC-derived virus via the intravenous (i.v.) route. We used this animal model to establish the ability to infect marmosets with bacterial artificial chromosome (BAC)-derived KSHV and to establish the role of individual glycoproteins (GPs) in the colonization of specific host tissues. We infected three groups of four animals each with BAC16-derived KSHV wild type (wt), KSHV gHASAELAAN, or KSHV{Delta}K8.1. KSHV gHASAELAAN contains mutations in gH that interfere with binding to the EphA2 receptor, which normally forms a high-affinity complex with gH/gL, whereas KSHV{Delta}K8.1 does not express GP K8.1. The KSHV gHASAELAAN mutant was used at a relatively higher concentration to overcome its intrinsic defect and to allow for analysis of tissue tropism. While seroconversion to the KSHV LANA antigen was variable, all animals exhibited clearly detectable viral DNA load in many tissues. Highest loads were detected in spleen, liver, heart and bone marrow. Viral DNA loads in these four tissues did not significantly differ between groups infected with KSHV wt or with the gHASAELAAN mutant. However, KSHV{Delta}K8.1-infected animals exhibited significantly lower levels of viral DNA in liver and bone marrow, but not in spleen or heart. Analysis of immune cells demonstrated significant activation of CD4+ and CD8+ T-cells in KSHV wt-infected animals. In wt and gHASAELAAN-infected animals, different B cell subpopulations were found to expand after infection, which implies that the gH/gL glycoprotein complex may shape the host response to KSHV in unexpected ways. In summary, our findings demonstrate that neither the interaction with Eph family receptors nor GP K8.1 is essential for infection via the i.v. route but K8.1 plays a critical role for KSHV tissue tropism in vivo.

microbiology↗

bioRxiv2023

Kaposi's sarcoma-associated herpesvirus (KSHV) gB dictates a low-pH endocytotic entry pathway as revealed by a dual-fluorescent virus system and a rhesus monkey rhadinovirus expressing KSHV gB

Interaction with host cell receptors initiates internalization of Kaposis sarcoma-associated herpesvirus (KSHV) particles via endocytosis or macropinocytosis. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of gH, gL, and gB, that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed single- and dual-fluorescent recombinant KSHV strains that incorporate tagged glycoproteins, capsid proteins or a combination thereof. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. Our data demonstrate lytic reactivation and infectivity of dual-fluorescent KSHV and incorporation of fluorescently-tagged proteins into viral particles. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. By using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established the KSHV fusion kinetics upon infection of different target cells. We measured marked differences in the "time-to-fusion" between different cell types. Inhibition of vesicle acidification prevented virus-cell fusion, implicating low vesicle pH as a requirement for the KSHV fusion step. These findings were corroborated by comparison of RRV-YFP reporter virus with wildtype gB and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB, these two novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion.

microbiology↗

bioRxiv2023

Tim-1 and Tim-4 mediate entry of the human Kaposi's sarcoma-associated herpesvirus and the related rhesus monkey rhadinovirus

Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus. It is associated with Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. KSHV is known to interact with several different receptors, among them heparan sulfate proteoglycans, Eph family receptors, and integrins. We mutated the closely related rhesus monkey rhadinovirus in the known receptor interaction sites for Eph family and Plexin domain containing proteins and found it to still replicate on certain cells. This lytic virus was then used as a selection agent in a genome-wide CRISPR knockout screen, which identified TIM1 and NRP1 as host dependency factors. NRP1 is also host factor for the related Epstein-Barr virus and was recently reported to promote KSHV infection, which we confirm even if it functions with low efficiency on most cells and became functional only after ablation of the Eph receptor interaction. Further analysis through overexpression demonstrated that Tim-1 and the related Tim-4 are strong mediators of RRV and KSHV infection, in particular in the absence of other receptor interactions and even more pronounced for a KSHV mutant deleted in glycoprotein K8.1. Both Tim-1 and Tim-4 are heavily O-glycosylated phosphatidylserine (PS) receptors. For KSHV in particular, experiments with mutated Tim-1 and comparison to Ebola virus glycoprotein-driven entry indicate that the interaction with Tim-1 occurs through PS-binding by Tim-1 and suggest additional interaction in a PS-independent manner. The mucin-like domain of Tim-1 is required for optimal receptor function. The use of Tim proteins for entry is a novelty for herpesviruses and underscores the unique biology of KSHV and RRV.

microbiology↗

bioRxiv2023

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes

Kaposis sarcoma-associated herpesvirus (KSHV) is associated with Kaposis sarcoma and several B cell malignancies. K8.1, the major antigenic component of the KSHV virion, has been reported to play a critical role in the infection of certain B cells, but otherwise its function remains enigmatic. We created a K8.1 knockout virus (KSHV{Delta}K8.1) in the BAC16 genetic background and analyzed its infectivity on a range of adherent cells. We observed a strong defect on several epithelial cells, e.g. the HaCaT keratinocyte model cell line, HEK 293T and A549 lung epithelial cells, but no such defect on other cells, among them e.g. lymphatic and blood endothelial cells. Mechanistically, we found that reduced infectivity of the K8.1 knockout virus correlated with reduced attachment to HaCaT cells. The defect in infectivity of KSHV{Delta}K8.1 could be rescued by complementation through expression of K8.1 in KSHV{Delta}K8.1 producing cells by means of a lentiviral vector. In a coculture infection model, KSHV{Delta}K8.1 was highly efficient at infecting the BJAB B cell line but was significantly impaired at infecting the MC116 B cell line, in line with a previous report. In fusion assays together with the gH/gL glycoprotein complex and gB, the components of the conserved herpesviral core fusion machinery, we did not observe activation of membrane fusion by K8.1 or its R8.1 homolog of the rhesus monkey rhadinovirus. In summary, we found K8.1 to function in a highly cell-specific manner during KSHV entry at the attachment step, playing an important role in the infection of epithelial cells.

microbiology↗

bioRxiv2021

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposis Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell Type-Specific Manner.

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma2-herpesviruses Kaposis sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549, human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell type-dependent inhibitors of KSHV and RRV infection in A549 and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell type-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. IMPORTANCEIFITM proteins are the first line of defense against infection by many pathogens, which may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action are well understood. We report here that in some cells, but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV, and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

microbiology↗

bioRxiv2020

SARS-CoV-2 differs from SARS-CoV in the requirements for receptor expression and proteolytic activation to trigger cell-cell fusion and is not inhibited by Bromhexine

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCECell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

microbiology↗

bioRxiv2019

EphA7 functions as a receptor for cell-to-cell transmission of Kaposi’s sarcoma-associated herpesvirus into BJAB B cells and for cell-free virus infection by the related rhesus monkey rhadinovirus

Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma and is associated with two B cell malignancies, primary effusion lymphoma and the plasmablastic variant of multicentric Castlemans disease. EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV for several adherent cell types. KSHV gH/gL was previously shown to also weakly interact with other members of the Eph family of receptors. Whether these interactions are of functional consequence remained unclear so far, even if other A-type Ephs were shown to be able to compensate for absence of EphA2 in overexpression systems. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells by over 80%. Finally, we demonstrate that receptor function of EphA7 is conserved between cell-to-cell transmission of KSHV and cell-free infection by the related rhesus monkey rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells.\n\nIMPORTANCEInfection of B cells is biologically relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castlemans disease (MCD) and primary effusion lymphoma (PEL). Elucidating the process of B cell infection is therefore important to understand the pathogenesis of these diseases. For various types of adherent cells, EphA2 has been shown by several groups to function as an entry receptor that is engaged by the gH/gL glycoprotein complex. Our previous findings indicate that KSHV does not only interact with EphA2, but can also bind to other members of the Eph family of receptor tyrosine kinases with comparatively lower avidity. We now analyzed the requirement of Eph interactions for infection of the BJAB B cell line, a model for infection of B cells by KSHV. We identify EphA7 as the principal Eph receptor for infection of this model B cell line by both KSHV and the related rhesus monkey rhadinovirus.

microbiology↗