Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Spatial tumour gene signature discriminates neoplastic from non-neoplastic compartments in colon cancer: unravelling predictive biomarkers for relapse.

BACKGROUND: Opting for or against the administration of adjuvant chemotherapy in therapeutic management of stage II colon cancer remains challenging. Several studies report few survival benefits for patients treated with adjuvant therapy and additionally revealing potential side effects of overtreatment, including unnecessary exposure to chemotherapy-induced toxicities and reduced quality of life. Predictive biomarkers are urgently needed. We, therefore, hypothesise that the spatial tissue composition of relapsed and non-relapsed colon cancer stage II patients reveals relevant biomarkers. METHODS: The spatial tissue composition of stage II colon cancer patients was examined by a novel spatial transcriptomics technology with sub-cellular resolution, namely in situ sequencing. A panel of 176 genes investigating specific cancer-associated processes such as apoptosis, proliferation, angiogenesis, stemness, oxidative stress, hypoxia, invasion and components of the tumour microenvironment was designed to examine differentially expressed genes in tissue of relapsed versus non-relapsed patients. Therefore, FFPE slides of 10 colon cancer stage II patients either classified as relapsed (5 patients) or non-relapsed (5 patients) were in situ sequenced and computationally analysed. RESULTS: We identified a tumour gene signature that enables the subclassification of tissue into neoplastic and non-neoplastic compartments based on spatial expression patterns obtained through in situ sequencing. We developed a computational tool called Genes-To-Count (GTC), which automates the quantification of in situ signals, accurately mapping their position onto the spatial tissue map and automatically identifies neoplastic and non-neoplastic tissue compartments. The GTC tool was used to quantify gene expression of biological processes upregulated within the neoplastic tissue in comparison to non-neoplastic tissue and within relapsed versus non-relapsed stage II colon patients. Three differentially expressed genes (FGFR2, MMP11 and OTOP2) in the neoplastic tissue compartments of relapsed patients in comparison to non-relapsed patients were identified predicting recurrence in stage II colon cancer. CONCLUSIONS: In depth spatial in situ sequencing showed potential to provide a deeper understanding of the underlying mechanisms involved in the recurrence of disease and revealed novel potential predictive biomarkers for disease relapse in colon cancer stage II patients. Our open-access GTC-tool allowed us to accurately capture the tumour compartment and quantify spatial gene expression in colon cancer tissue.

Influenza A virus strain PR/8/34, but neither HAM/2009 nor WSN/33, is transiently inhibited by the PB2-targeting drug paliperidone.

Influenza A virus (FLUAV) is a significant human pathogen. In silico structural analysis (PMID 28628827) has suggested that the FDA-approved drug paliperidone interferes with the binding of the FLUAV polymerase subunit PB2 to the nucleoprotein NP. We found that paliperidone inhibits FLUAV A/PR/8/34 early after infection of canine MDCK II, human A549, and human primary bronchial cells, but not at late time points. No effect was detectable against the strains A/Hamburg/05/2009 and A/WSN/33. Moreover, paliperidone indeed disturbed the interaction between the PB2 and the NP of A/PR/8/34 and reduced early viral RNA and protein synthesis by approximately 50%. Thus, paliperidone has measurable but transient and virus-strain-restricted effects on FLUAV.

Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs.

Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.

Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks.

Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its nonadapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism, and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases, including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, these data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation; on the other hand, the host cell triggers antiviral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This study revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, we identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2α inhibitor.

Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells.

BACKGROUND: Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. METHODS: To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. RESULTS: We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. CONCLUSION: Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

Influenza virus-induced caspase-dependent enlargement of nuclear pores promotes nuclear export of viral ribonucleoprotein complexes.

UNLABELLED: Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCE: In contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.

[Coping of cybervictimization in adolescence - emotional and behavioral reactions to cyberbullying ]. / Bewältigung von Cyberviktimisierung im Jugendalter - Emotionale und verhaltensbezogene Reaktionen auf Cyberbullying.

The experience of cybervictimization is related to health, psychological, and behavioral problems among children and adolescents. Up to today research is scarce, how the persons affected by cybervictimization react and which determinants influence the choice for social, problem-focused, technical, or helpless coping behavior. The current online study with 428 adolescents considers age, sex, mean internet use, frequency of victimization, roles in cyberbullying, and emotional reactions to cybervictimization as potential determinants of the mentioned coping strategies. Based on the participant role approach, roles of cyberbullies, cybervictims, defenders or outsiders are frequently changing. Logistic regression analyses point out the important relevance of emotional reactions like anger or helplessness and the roles as cyberbully-victim or outsider. Further, younger participants reported cybervictimization more often, while the frequency of cybervictimization and sex did not and internet use only partially predict coping strategies. These findings corroborate the relevance of emotional reactions and the roles in the process of cyberbullying. As a starting point for prevention and intervention of cybervictimization, we suggest emotion regulation, teaching of technical coping behaviors as well as reflexion of roles in the context of cyberbullying. If feasible, different stakeholders should be engaged in this process: adolescents, parents, educational staff inside and outside of schools, experts from counseling and therapy as well as internet and mobile phone service providers.

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1-10 µM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1-12 µM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Extracapsular extension of pN2 lymph node metastases is not prognostically significant in surgically resected patients with non-small cell lung cancer.

Objectives: In patients with non-small cell lung cancer (NSCLC) the pathologic lymph node status N2 is a heterogeneous entity, with different degrees of lymph node involvement representing different prognoses. It is speculated whether extra capsular nodal extension may help to define a subgroup with implications on long-term survival. Methods: We retrospectively identified 118 patients with non-small cell lung cancer (65 men, 53 women), who were treated between 2013 and 2018 and found to have pathologic N2 lymph node involvement. In all patients lung resection with systematic mediastinal and hilar lymph node dissection was performed with curative intent. In N2 lymph node metastases capsules of affected lymph nodes were examined microscopically as to whether extracapsular extension was present. Results: 51 patients (43 %) had extracapsular extension (ENE). Most of these patients (n=35) only had ENE in a single lymph node (69 %). The overall 5-year survival rate was 24.6 % and progression-free survival rate 17.8 %. In the multivariate analysis OS was worse for patients with multiple affected pN2 stations, concurrent N1 metastases, increasing age, and larger tumor size. For the percentage of lymph nodes affected with ENE (of total examined) only a non-significant trend towards worse OS could be observed (p=0.06). Conclusions: Although we could not demonstrate significant prognostic differences between N2 extra capsular nodal involvement within our patient population, other analyses may yield different results. However, clinicians should continue performing thorough lymph nodes dissections in order to achieve local complete resection even in patients with extra capsular tumor spread.

Structure-based lead optimization of peptide-based vinyl methyl ketones as SARS-CoV-2 main protease inhibitors.

Despite several major achievements in the development of vaccines and antivirals, the fight against SARS-CoV-2 and the health problems accompanying COVID-19 are still ongoing. SARS-CoV-2 main protease (Mpro), an essential viral cysteine protease, is a crucial target for the development of antiviral agents. A virtual screening analysis of in-house cysteine protease inhibitors against SARS-CoV-2 Mpro allowed us to identify two hits (i.e., 1 and 2) bearing a methyl vinyl ketone warhead. Starting from these compounds, we herein report the development of Michael acceptors targeting SARS-CoV-2 Mpro, which differ from each other for the warhead and for the amino acids at the P2 site. The most promising vinyl methyl ketone-containing analogs showed sub-micromolar activity against the viral protease. SPR38, SPR39, and SPR41 were fully characterized, and additional inhibitory properties towards hCatL, which plays a key role in the virus entry into host cells, were observed. SPR39 and SPR41 exhibited single-digit micromolar EC50 values in a SARS-CoV-2 infection model in cell culture.

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection.

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1-3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein-protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5'-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5'-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

Differential Impact of Specific Amino Acid Residues on the Characteristics of Avian Influenza Viruses in Mammalian Systems.

Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1EGY, H9N2EGY, H5N1PB2-H9N2EGY, H5N1H9N2_PB2_K591Q, H5N1PB2_K627E, H5N1PB2_K627E/591K, H5N1PB2_627K/591K). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1PB2_K627E/591K, H5N1PB2_627K/591K) and the reassortant H5N1PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1H9N2_PB2_K591Q, H5N1PB2_K627E) replicated to significantly lower levels than the parental H5N1EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1EGY and H9N2EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro.

Targeting the DEAD-Box RNA Helicase eIF4A with Rocaglates-A Pan-Antiviral Strategy for Minimizing the Impact of Future RNA Virus Pandemics.

The increase in pandemics caused by RNA viruses of zoonotic origin highlights the urgent need for broad-spectrum antivirals against novel and re-emerging RNA viruses. Broad-spectrum antivirals could be deployed as first-line interventions during an outbreak while virus-specific drugs and vaccines are developed and rolled out. Viruses depend on the host's protein synthesis machinery for replication. Several natural compounds that target the cellular DEAD-box RNA helicase eIF4A, a key component of the eukaryotic translation initiation complex eIF4F, have emerged as potential broad-spectrum antivirals. Rocaglates, a group of flavaglines of plant origin that clamp mRNAs with highly structured 5' untranslated regions (5'UTRs) onto the surface of eIF4A through specific stacking interactions, exhibit the largest selectivity and potential therapeutic indices among all known eIF4A inhibitors. Their unique mechanism of action limits the inhibitory effect of rocaglates to the translation of eIF4A-dependent viral mRNAs and a minor fraction of host mRNAs exhibiting stable RNA secondary structures and/or polypurine sequence stretches in their 5'UTRs, resulting in minimal potential toxic side effects. Maintaining a favorable safety profile while inducing efficient inhibition of a broad spectrum of RNA viruses makes rocaglates into primary candidates for further development as pan-antiviral therapeutics.

Benzene degradation in contaminated aquifers: Enhancing natural attenuation by injecting nitrate.

Natural attenuation processes depend on the availability of suitable electron acceptors. At the megasite Zeitz, concentrations of the main contaminant benzene were observed to increase constantly in the lower aquifer to levels of more than 2.5 mM. This was accompanied by decreasing concentrations of sulphate (SO42-), which has been previously shown to be the main electron acceptor for benzene oxidation at this site, resulting in an electron acceptor-limited, sulphidic benzene plume. Therefore, a field experiment was conducted to stimulate benzene biodegradation by injecting nitrate (NO3-) into the sulphidic benzene plume aiming (i) to recycle sulphate by nitrate-dependent sulphide oxidation, and (ii) to serve as direct electron acceptor for benzene oxidation. Within 60 days, 6.74 tons sodium nitrate (NaNO3) were injected into the lower aquifer, and the resulting biogeochemical effects within the benzene plume were monitored for more than one year by chemical and microbiological analyses of groundwater samples taken from various depths of ten monitoring wells located in three observation lines downstream of nitrate injection. Nitrate was microbiologically consumed, as shown by changes in δ15N-NO3- and δ18O-NO3- values, partial nitrite accumulation, and changing ratios of Na+/NO3-. Main electron donors for nitrate reduction were reduced sulphur compounds, verified by changing δ34S-SO42- and δ18O-SO42- values, partially increasing sulphate concentrations, and strongly increasing abundances of typical sulphur-oxidizing, nitrate-reducing bacterial taxa within the nitrate plume. The general absent hydrogen isotope fractionation of benzene, also in the sulphidic, nitrate-free part of the plume, indicates that benzene was not biodegraded by sulphate-reducing consortia. However, detected small carbon isotope fractionation of benzene points to in situ benzene biodegradation processes in the plume, probably supported by nitrate. In conclusion, nitrate injection resulted in changing redox conditions and recycling of sulphate in the sulphidic, sulphate-depleted benzene plume due to microbial oxidation of reduced sulphur species, leading to presumably favored conditions for in situ benzene biodegradation.

The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (-) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2.

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PLpro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PLpro . Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PLpro are valuable starting points for the development of new pan-coronaviral inhibitors.

Multi-level inhibition of coronavirus replication by chemical ER stress.

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.