Biology subjects

Ensser, A.

Publications and source records attributed to Ensser, A..

15 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↗

bioRxiv2024

SARS-CoV-2 Omicron Envelope T9I adaptation confers resistance to autophagy

To date, five variants of concern (VOCs) of SARS-CoV-2 have emerged that show increased fitness and/or immune evasion. While the continuously evolving escape from humoral immune responses has been analyzed in detail, adaptation of SARS-CoV-2 to human innate immune defenses such as autophagy is less understood. Here, we demonstrate that mutation T9I in the structural envelope (E) protein confers autophagy resistance of Omicron VOCs (BA.1, BA.5 and XBB.1.5) compared to 2020 SARS-CoV-2 or the Delta VOC. Mechanistic analyses revealed that Omicron-associated E T9I shows increased inhibition of autophagic flux and colocalization/interaction with autophagosomes, thus shielding incoming SARS-CoV-2 S pseudotyped virions from autophagy. Rare Omicron isolates carrying ancestral E T9 remain sensitive towards autophagy whereas recombinant early 2020 SARS-CoV-2 expressing E T9I shows increases resistance against autophagy. Our data indicate that the E T9I mutation drives autophagy resistance of the Omicron variants and thus may have contributed to their effective spread.

microbiology↗

bioRxiv2024

DNA virus infections shape transposable elements activity in vitro and in vivo

Transposable elements (TEs) are implicated in a variety of processes including placental and preimplantation development and a variety of human diseases. TEs are known to be activated in the context of some viral infections, but the mechanisms and consequences are not understood. We show strong activation of TEs upon DNA virus infection, in particular the MLT- and THE1-class of LTR-containing retrotransposons as well as a subset of LINE-1-, Alu-elements and HERVs. Mechanistically, two key pathways induce TEs upregulation: inhibition of the KAP1/TRIM28 repressive complex by phosphorylation, and expression of the pioneer transcription factor double-homeobox 4 (DUX4), which is known to be involved in TE-induction during zygotic genome activation in embryonic development. DUX4 is induced by DNA viruses, it binds to TEs upon infection and analysis of genes adjacent to TEs shows pathways that are important for DNA virus infections. Analysis of knockdown, knockout and overexpression data reveal that almost all TEs expressed upon herpesviral infection are regulated by KAP1/TRIM28 and DUX4. Interestingly, analysis of single cell sequencing data from patients with DNA virus-associated cancers showed that in vivo TEs expression strongly correlates with virus infection, indicating a possible role in viral oncogenesis.

genomics↗

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↗

bioRxiv2023

SARS-CoV-2 ORF3c suppresses immune activation by inhibiting innate sensing

SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While most of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. For example, the ORF3a sgRNA also encodes ORF3c, an enigmatic 4l-amino acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2 infected cells and suppresses RIG-I- and MDA5-mediated immune activation and IFN-{beta} induction. Mechanistic analyses revealed that ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c demonstrating that this reading frame is not essential for efficient viral replication in vivo and likely compensated by other viral proteins. In agreement with this, disruption of ORF3c did not significantly affect SARS-CoV-2 replication in CaCo-2 or CaLu-3 cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

microbiology↗

bioRxiv2022

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

microbiology↗

bioRxiv2022

SARS-CoV-2 variants show temperature-dependent enhancedpolymerase activity in the upper respiratory tract and high transmissibility

With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33{degrees}C) and lower (37{degrees}C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33{degrees}C compared to 37{degrees}C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33{degrees}C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.

microbiology↗

bioRxiv2021

Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization

Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

immunology↗

bioRxiv2021

Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

microbiology↗

bioRxiv2021

A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly generate human monoclonal antibodies. After immunizing these mice against the spike protein of SARS-CoV-2, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralized SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of neutralizing antibodies binds to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2 induced weight loss. Thus, we report two clusters of potent non-competing SARS-CoV-2 neutralizing antibodies providing potential candidates for therapy and prophylaxis of COVID-19. The study further supports the use of transgenic animals with human immunoglobulin gene repertoires in pandemic preparedness initiatives.

immunology↗

bioRxiv2021

Herpesviral induction of germline transcription factor DUX4 is critical for viral gene expression

DUX4 is a transcription factor and a master regulator of embryonic genome activation (EGA). During early embryogenesis, EGA is crucial for maternal to zygotic transition at the 8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In adult somatic cells, DUX4 expression is largely silenced. Activation is likely pathogenic, and in adult muscle cells causes genetic disorder Facioscapulohumeral Muscular Dystrophy (FSHD). We identified activation of DUX4 expression upon lytic replication of the herpesviruses HSV-1, HCMV, EBV and KSHV, but not of adenoviruses, negative strand RNA viruses or positive strand RNA viruses. We demonstrate by RNA-Seq analysis that DUX4 expression upon herpesviral replication leads to the induction of hundreds of DUX4 target genes including germline-specific retroelements as well as several members of the TRIM, PRAMEF and ZSCAN protein families. Moreover, we show that DUX4 expression is a direct consequence of herpesviral infection. DUX4 can be stimulated by overexpression of HSV-1 immediate early proteins, indicating active induction of EGA genes by herpesviral infection. We further show that DUX4 expression is critical for driving HSV-1 gene expression. Our results show that viruses from alpha-, beta- and gamma-herpesvirus subfamilies induce DUX4 expression and downstream germline-specific genes and retroelements. We hypothesize that herpesviruses induce DUX4 expression in order to induce an early embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.

microbiology↗

bioRxiv2020

CRNKL1 is a highly selective regulator of intron-retaining HIV-1 and cellular mRNAs

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely-spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knock-down. Depletion of DHX38, WDR70 and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of cellular mRNAs with selectively retained introns. Thus, CRNKL1 dependent nuclear retention seems to be a novel mechanism for the regulation of cytoplasmic levels of intron-retaining cellular mRNAs that is harnessed by HIV-1 to direct its complex splicing pattern.

molecular biology↗

bioRxiv2020

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV)

The rhesus monkey rhadinovirus (RRV), a {gamma}2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposis sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by approx. 60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 in in vitro assays and reduced RRV infection in a cell-type specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV. AUTHORS SUMMARYKSHV is the causative agent of a group of malignancies which account for a substantial disease burden in particular in sub-Saharan Africa. RRV, a related virus of rhesus macaques, has shown promise as model system for KSHV and for the development of immunization strategies. To exploit the full potential of the RRV animal model system, detailed knowledge of commonalities and differences between KSHV and RRV is key. Here, we describe the Plexin domain containing proteins 1 and 2 as a novel receptor family which mediates entry of RRV, but not of KSHV. Infection experiments using RRV mutants deleted of the Plxdc interaction motif suggest a cell type-specific contribution of Plxdc receptors to RRV infection. As information on Plxdc1/2 and its biological function is still sparse, analysis of the RRV-Plxdc interaction will help to characterize the physiological and pathophysiological role of this receptor family.

microbiology↗