Biology subjects

Widera, M.

Publications and source records attributed to Widera, M..

17 recordsLinked to original sources

bioRxiv2023

Cellular hnRNPA0 limits HIV-1 production by interference with LTR-activity and programmed ribosomal frame-shifting

The interplay between host factors and viral components has a profound impact on the viral replication efficiency and fitness. Heterogeneous nuclear ribonucleoproteins (hnRNPs), in particular members of the subfamily A/B, have been broadly studied as HIV-1 host dependency factors, however, the least related member hnRNPA0 has so far not been functionally studied in its potential role affecting viral replication. In this study, we revealed that hnRNPA0 overexpression in HEK293T cells significantly reduced HIV-1 long terminal repeat (LTR) activity up to 3.5-fold, leading to a significant decrease in total viral mRNA (5.5-fold) and protein levels (3-fold). Conversely, knockdown of hnRNPA0 enhanced LTR activity, suggesting its negative regulatory role in viral gene expression. Moreover, the splicing pattern of HIV-1 remained largely unaffected by altered hnRNPA0 levels indicating changes in viral mRNA expression predominantly occurred at the transcriptional level. Moreover, hnRNPA0 overexpression was found to significantly reduce the programmed ribosomal frameshift efficiency of HIV-1, resulting in a shift in the HIV-1 p55/p15 ratio, compromising viral fitness. Synergistic inhibition of LTR activity and thus reduced viral mRNA transcription and impaired ribosomal frameshifting efficiency, which is important for viral infectivity, were detrimental to HIV-1 replication. Additionally, our study revealed that hnRNPA0 levels were lower in therapy naive HIV-1-infected individuals compared to healthy controls and temporarily repressed after IFN-I treatment in HIV-1 target cells. Our findings highlight the significant role of hnRNPA0 in HIV-1 replication and suggests that its IFN-I regulated expression levels are decisive for viral fitness. ImportanceRNA binding proteins, in particular heterogeneous nuclear ribonucleoproteins (hnRNPs) have been extensively studied as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. However, the functional role of hnRNPA0, the least related member of the hnRNPA/B family, and its potential impact on viral replication remains unclear. For the first time, our findings demonstrate the significance of hnRNPA0 in restricting viral replication efficiency. We demonstrate that hnRNPA0 plays a pleiotropic role in limiting viral replication being a negative regulator of viral transcription and significantly impairing ribosomal frameshifting. Our study also revealed hnRNPA0 as an IFN-regulated host factor that is temporarily repressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naive HIV-1-infected individuals compared to healthy controls. Understanding the mode of action between hnRNPA0 and HIV-1 might help to identify novel therapeutically strategies against HIV-1 and other viruses.

microbiology↗

bioRxiv2022

TMPRSS2 is essential for SARS-CoV-2 Beta and Omicron infection

The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts whether TMPRSS2 inhibitors would be suitable for treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to spread of SARS-CoV-2 in the infected host is largely unclear. Here, we show that loss of TMPRSS2 strongly reduced the replication of the Beta variant in nose, trachea and lung of C57BL mice and protected the animals from weight loss and disease. Infection of mice with the Omicron variant did not cause disease, as expected, but again TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify a key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection and highlight TMPRSS2 as an attractive target for antiviral intervention.

immunology↗

bioRxiv2022

Long-term immune protection against SARS-CoV-2 escape variants upon a single vaccination with murine cytomegalovirus expressing the spike protein

Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have led to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMV) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant murine CMV (MCMV) vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, S-specific humoral and cellular immunity was not only maintained but even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two immune-evading variants of concern (VoCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19. Authors SummaryWhile widespread vaccination has substantially reduced risks of severe COVID presentations and morbidity, immune waning and continuous immune escape of novel SARS-CoV-2 variants have resulted in a need for numerous vaccine boosters and a continuous adaptation of vaccines to new SARS-CoV-2 variants. We show in proof of principle experiments with a recombinant murine cytomegalovirus expressing the SARS-CoV-2 spike protein (MCMVS) that one immunization with a CMV vaccine vector drives enduring protection in both young and aged mice, with long-term maturation of immune responses that broaden the antiviral effects over time. Hence, this approach resolves issues of immune waning and mitigates the effects of COVID-19 evolution and immune escape, reducing the need for additional immunizations and potentially improving vaccine compliance.

immunology↗

bioRxiv2022

SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

microbiology↗

bioRxiv2022

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype

Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in naive hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naive K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

microbiology↗

bioRxiv2022

Low-dose bivalent mRNA vaccine is highly effective against different SARS-CoV-2 variants in a transgenic mouse model

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve COVID-19 control. We compared monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein, primarily in a transgenic mouse model and a Wistar rat model. The low-dose bivalent mRNA vaccine contained half the mRNA of each respective monovalent vaccine, but induced comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, specific CD4+ and CD8+ responses, and fully protected transgenic mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduced viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized Wistar rats also contained neutralizing antibodies against the B.1.1.529 (Omicron BA.1) variant. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins is a feasible approach for increasing the potency of vaccines against emerging and co-circulating SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Omicron variant of SARS-CoV-2 exhibits an increased resilience to the antiviral type I interferon response

The new variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype strain (B.1) with respect to their interactions with the antiviral type I interferon (IFN-alpha/beta) response in infected cells. Our data indicate that Omicron has gained an elevated capability to suppress IFN-beta induction upon infection and to better withstand the antiviral state imposed by exogenously added IFN-alpha.

microbiology↗

bioRxiv2022

Reduced interferon antagonism but similar drug sensitivity in Omicron variant compared to Delta variant SARS-CoV-2 isolates

The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses.

microbiology↗

bioRxiv2021

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

microbiology↗

bioRxiv2021

Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

Efficient replication of HIV-1 depends on balanced levels of host cell components, including cellular splicing factors. Type I interferons (IFN-I), playing a crucial role in the innate immune defense against viral infections, are well known to induce the transcription of IFN-stimulated genes (ISGs) including potent host restriction factors. Not so well known is, that IFN-repressed genes (IRepGs) also affect viral infections by downregulating host dependency factors that are essential for viral replication. So far, knowledge about IRepGs involved in HIV-1 infection is very limited. Here, we demonstrate that expression levels of the serine/arginine-rich splicing factor 1 (SRSF1) were repressed upon treatment with IFN subtypes in HIV-1 susceptible cell lines as well as primary cells. Furthermore, we could demonstrate in two independent patient cohorts that HIV-1 infection and the concomitant inflammation during the acute and chronic phase, resulted in the strong induction of ISGs, but at the same time significantly repressed SRSF1. 4sU-labeling of newly transcribed mRNAs revealed that IFN-mediated repression of SRSF1 originated from a transcriptional shutdown. Experimental downregulation as well as overexpression of SRSF1 expression levels resulted in crucial changes in HIV-1 LTR-transcription, alternative splice site usage and virus production. While lower SRSF1 levels resulted in low vif mRNA levels and thus severely reduced viral infectivity, higher levels of SRSF1 impaired LTR-Tat-activity and HIV-1 particle production. Our data highlight the so far undescribed role of SRSF1 acting as an IFN-repressed cellular dependency factor decisively regulating HIV-1 post integration steps. Author SummaryIFN-I play a central role in the innate immune defense against viral infections by regulating the expression of interferon stimulated genes (ISGs) and interferon repressed genes (IRepGs). The stimulation of host restriction factors and the reduction of host dependency factors decisively affects the efficiency of HIV-1 replication. After the stable integration of the provirus into the host chromosome, HIV-1 exploits the host cell transcription and splicing machinery for its replication. A network of conserved splice sites and splicing regulatory elements maintain balanced levels of viral transcripts essential for virus production and immune evasion. We demonstrate the so far undescribed role of the splicing factor SRSF1 as an IRepG crucially involved in HIV-1 RNA processing. In HIV-1 infected individuals, we observed inversely proportional expression of high ISG15 and low SRSF1 levels, which were restored in ART treated patients. We could demonstrate, that IFN-I stimulation of HIV-1 target cells resulted in a significant repression of SRSF1 RNA and protein levels. Since low SRSF1 expression decisively reduced HIV-1 vif mRNA levels, a severe impairment of viral replication was observed in APOBEC3G expressing cells. As overexpression negatively affected HIV-1 LTR transcription and virus production, balanced levels of SRSF1 are indispensable for efficient replication.

microbiology↗

bioRxiv2021

Intranasal administration of a monoclonal neutralizing antibody protects mice against SARS-CoV-2 infection

Despite recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both, prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2 and retains activity against the variants of concern B.1.1.7 and B.1.351. Importantly, not only systemic but also intranasal application of DZIF-10c abolished presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies. Significance StatementMonoclonal neutralizing antibodies are important in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection. However, their intravenous application might lead to suboptimal bioavailability in the lung. We here precisely characterize a new monoclonal neutralizing antibody (DZIF-10c) that binds to the receptor binding domain of the spike protein of SARS-CoV-2. DZIF-10c neutralizes SARS-CoV-2 with exceptionally high potency and maintains activity against circulating variants of concern. The antibody has a favorable pharmacokinetic profile and protects mice from SARS-CoV-2 infection. Importantly, we show that intranasal administration of DZIF-10c generates protective efficacy. These results not only identify DZIF-10c as a novel highly potent neutralizing antibody, but further pave the way for a topical application of anti-SARS-CoV-2 antibodies.

microbiology↗

bioRxiv2021

Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

microbiology↗

bioRxiv2021

The CCL2 Chemokine Promotes Early Seeding of the Latent HIV Reservoir

HIV infects long-lived CD4 memory T cells establishing a latent viral reservoir that necessitates lifelong anti-retroviral therapy (ART). How this reservoir is formed so swiftly remains unknown. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, which can drive recruitment of cells expressing the CCR2 receptor including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find 1) treatment of humanized mice with anti-CCL2 antibodies during HIV infection decreases reservoir seeding and 2) CCR2/5+ cells from the blood of HIV-infected individuals on long term ART contain significantly more provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the hosts innate inflammatory CCL2 response to HIV infection recruits CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation likely contributing to rapid formation of the latent HIV reservoir. GRAPHICAL ABSTRACTWhy is the latent HIV reservoir established so early following infection? An innate immune response occurs during acute infection that establishes a "zone of inflammation" (step 1). The CCL2 chemokine is produced in part through IFI16 sensing of HIV DNA in abortively infected cells. CCL2 promotes rapid recruitment of CCR2/5+ memory CD4 T cells (step 2). Many of these cells become productively infected (step 3) and a fraction become latently infected (step 4). Thus, HIV hijacks the host inflammatory response to rapidly establish the latent reservoir. In support of this model, we find HIV reservoir reduction in humanized mice treated with anti-CCL2 antibodies during early infection. Further, we find that CCR2/5+ CD4 T cells harbor a substantial fraction of detectable proviruses in the blood of HIV-infected individuals on long-term suppressive ART. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/432826v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@8d6da8org.highwire.dtl.DTLVardef@11a3a1corg.highwire.dtl.DTLVardef@dd05dborg.highwire.dtl.DTLVardef@ab84e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, the stability of the virus in cell culture media at 4{degrees}C and on touch panel surfaces used in laboratory environment was analyzed. In addition, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.

microbiology↗

bioRxiv2020

Optimized qRT-PCR approach for the detection of intra- and extracellular SARS-CoV-2 RNAs

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

microbiology↗

bioRxiv2020

SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles

SARS-CoV-2 is a novel coronavirus currently causing a pandemic. We show that the majority of amino acid positions, which differ between SARS-CoV-2 and the closely related SARS-CoV, are differentially conserved suggesting differences in biological behaviour. In agreement, novel cell culture models revealed differences between the tropism of SARS-CoV-2 and SARS-CoV. Moreover, cellular ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (enables virus entry via S protein cleavage) levels did not reliably indicate cell susceptibility to SARS-CoV-2. SARS-CoV-2 and SARS-CoV further differed in their drug sensitivity profiles. Thus, only drug testing using SARS-CoV-2 reliably identifies therapy candidates. Therapeutic concentrations of the approved protease inhibitor aprotinin displayed anti-SARS-CoV-2 activity. The efficacy of aprotinin and of remdesivir (currently under clinical investigation against SARS-CoV-2) were further enhanced by therapeutic concentrations of the proton pump inhibitor omeprazole (aprotinin 2.7-fold, remdesivir 10-fold). Hence, our study has also identified anti-SARS-CoV-2 therapy candidates that can be readily tested in patients.

microbiology↗

bioRxiv2019

HIV infection does not alter Interferon α/β receptor expression on mucosal immune cells

The innate immune response induced by type I interferons (IFNs) play a critical role in the establishment of HIV infection. IFNs are induced early in HIV infection and trigger an antiviral defense program by signaling through the IFNa/b receptor (IFNAR), which consists of two subunits, IFNAR1 and IFNAR2. Changes in IFNAR expression in HIV target cells, as well as other immune cells, could therefore have important consequences for initial HIV spread. It was previously reported that IFNAR2 expression is increased in peripheral blood CD4+ CXCR4+ T cells of HIV+ patients compared to HIV uninfected controls, suggesting that HIV infection may alter the IFN responsiveness of target cells. However, the earliest immune cells affected by HIV in vivo reside in the gut-associated lymphoid tissue (GALT). To date, it remains unknown if IFNAR expression is altered in GALT immune cells in the context of HIV infection and exposure to IFNs, including the 12 IFNa subtypes. Here, we analyzed the expression of surface bound and soluble IFNAR2 on Lamina propria mononuclear cells (LPMCs) isolated from the GALT of HIV- individuals and in plasma samples of HIV+ patients. IFNAR2 expression varied between different T cells, B cells and natural killer cells, but was not altered following HIV infection. Furthermore, expression of the soluble IFNAR2a isoform was not changed in HIV+ patients compared to healthy donors, nor in LPMCs after HIV-1 infection ex vivo. Even though the 12 human IFN subtypes trigger different biological responses and vary in their affinity to both receptor subunits, stimulation of LPMCs with different recombinant IFN subtypes did not result in any significant changes in IFNAR2 surface expression. Our data suggests that potential changes in the IFN responsiveness of mucosal immune cells during HIV infection is unlikely dictated by changes in IFNAR2 expression.

immunology↗