Apalutamide Prevents SARS-CoV-2 Infection in Lung Epithelial Cells and in Human Nasal Epithelial Cells.

In early 2020, the novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, and rapidly propagated worldwide causing a global health emergency. SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) protein for cell entry, followed by proteolytic cleavage of the Spike (S) protein by the transmembrane serine protease 2 (TMPRSS2), allowing fusion of the viral and cellular membranes. Interestingly, TMPRSS2 is a key regulator in prostate cancer (PCa) progression which is regulated by androgen receptor (AR) signaling. Our hypothesis is that the AR signaling may regulate the expression of TMPRSS2 in human respiratory cells and thus influence the membrane fusion entry pathway of SARS-CoV-2. We show here that TMPRSS2 and AR are expressed in Calu-3 lung cells. In this cell line, TMPRSS2 expression is regulated by androgens. Finally, pre-treatment with anti-androgen drugs such as apalutamide significantly reduced SARS-CoV-2 entry and infection in Calu-3 lung cells but also in primary human nasal epithelial cells. Altogether, these data provide strong evidence to support the use of apalutamide as a treatment option for the PCa population vulnerable to severe COVID-19.

Desloratadine, an FDA-approved cationic amphiphilic drug, inhibits SARS-CoV-2 infection in cell culture and primary human nasal epithelial cells by blocking viral entry.

The 2019 global coronavirus (COVID-19) pandemic has brought the world to a grinding halt, highlighting the urgent need for therapeutic and preventive solutions to slow the spread of emerging viruses. The objective of this study was to assess the anti-SARS-CoV-2 effectiveness of 8 FDA-approved cationic amphiphilic drugs (CADs). SARS-CoV-2-infected Vero cells, Calu-3 cells and primary Human Nasal Epithelial Cells (HNEC) were used to investigate the effects of CADs and revealed their antiviral mode of action. Among the CADs tested, desloratadine, a commonly used antiallergic, well-tolerated with no major side effects, potently reduced the production of SARS-CoV-2 RNA in Vero-E6 cells. Interestingly, desloratadine was also effective against HCoV-229E and HCoV-OC43 showing that it possessed broad-spectrum anti-coronavirus activity. Investigation of its mode of action revealed that it targeted an early step of virus lifecycle and blocked SARS-CoV-2 entry through the endosomal pathway. Finally, the ex vivo kinetic of the antiviral effect of desloratadine was evaluated on primary Human Nasal Epithelial Cells (HNEC), showing a significant delay of viral RNA production with a maximal reduction reached after 72 h of treatment. Thus, this treatment could provide a substantial contribution to prophylaxis and systemic therapy of COVID-19 or other coronaviruses infections and requires further studies.

Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection.

Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu-sEVs) produced by human nasal epithelial cells (HNECs) in SARS-CoV-2 infection. We show that uninfected HNECs produce mu-sEVs containing SARS-CoV-2 receptor ACE2 and activated protease TMPRSS2. mu-sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu-sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS-CoV-2 virions prone to entry into target cells using the 'early', TMPRSS2-dependent pathway instead of the 'late', cathepsin-dependent route. These results indicate that prefusion Spike priming by mu-sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS-CoV-2 infection, but instead facilitates it.

Inhibition of SARS-CoV-2 Infection by the Cyclophilin Inhibitor Alisporivir (Debio 025).

Cyclophilins play a key role in the life cycle of coronaviruses. Alisporivir (Debio 025) is a nonimmunosuppressive analogue of cyclosporine with potent cyclophilin inhibition properties. Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner in Vero E6 cells, with a 50% effective concentration (EC50) of 0.46 ± 0.04 µM. Alisporivir inhibited a postentry step of the SARS-CoV-2 life cycle. These results justify rapidly conducting a proof-of-concept phase 2 trial with alisporivir in patients with SARS-CoV-2 infection.

Characterization of the Anti-Hepatitis C Virus Activity of New Nonpeptidic Small-Molecule Cyclophilin Inhibitors with the Potential for Broad Anti-Flaviviridae Activity.

Although members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC50s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.

Viruses and miRNAs: More Friends than Foes.

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host-pathogen interaction.

Low cross-neutralization of hepatitis C correlates with liver disease in immunocompromized patients.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection causes severe liver disease in HIV-infected patients and liver transplant recipients. The impact of serum and immunoglobulin on viral entry was analysed in these patients. METHOD: Sera from 60 anti-HCV positive patients, including 30 who were also anti-HIV positive, were tested with HCVpp from different genotypes (1a, 1b, 3 and 4) and with HCVcc (H77/JFH1). Seventeen HIV-seropositive and 13 HIV-seronegative patients with decompensated liver disease were studied before and after liver transplant. RESULTS: Serum neutralization was markedly lower after liver transplant and in HIV patients than in mono-infected immune-competent individuals. This effect was due to low antibody-mediated neutralization. In HIV patients, low neutralization was correlated with low lymphocyte T CD4 cell counts and the severity of liver disease. To characterize neutralization, we tested HCVpp lacking hypervariable region (HVR1) and SR-BI receptor cholesterol transfer inhibition by BLT-4. These experiments showed that neutralization was strongly dependent on the HVR1 and the SR-BI receptor. HVR1 sequences showed that selective pressures were low in immune-compromised patients and highly correlated to HCV neutralization after liver transplant. Neutralization experiments were reproduced with HCV strain JFH1. CONCLUSION: Serum neutralization in HIV-coinfected patients and HCV-infected liver transplant recipients is poor enhancing HCV entry through HVR1/SR-BI interplay. This may contribute to the severity of hepatitis C in these settings.

Human hepatic stellate cells are not permissive for hepatitis C virus entry and replication.

BACKGROUND: Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated. OBJECTIVE: To assess whether human HSCs are susceptible to HCV infection. DESIGN: We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs. RESULTS: In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication. CONCLUSIONS: Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process.

RNase Y is responsible for uncoupling the expression of translation factor IF3 from that of the ribosomal proteins L35 and L20 in Bacillus subtilis.

RNase Y is a novel endoribonuclease affecting global mRNA metabolism. We show that this nuclease affects the expression of the Bacillus subtilis infC-rpmI-rplT operon, encoding translation initiation factor IF3 and the ribosomal proteins L35 and L20. This operon is autoregulated by a complex L20-dependent transcription attenuation mechanism. L20 binds to a phylogenetically conserved domain on the 5' untranslated region of the infC mRNA which mimics the L20 binding sites on 23S rRNA. We have identified a second promoter (P1) upstream of the previously identified promoter (P2). The P1, but not the P2, readthrough transcript is stabilized in a strain depleted for RNase Y. However, under these conditions infC biosynthesis is repressed threefold. We show that the unprocessed P1 transcript is non-functional for IF3 translation but fully competent to express the co-transcribed ribosomal protein genes. RNase Y cleavage of the P1 transcript creates an entry site for the 5'-3' exonucleolytic activity of RNase J1 which degrades the infC mRNA when translation initiation efficiency is low. A second RNase Y cleavage is crucial for initiating degradation of the prematurely terminated infC leader RNAs, including the L20 operator complex, which permits efficient recycling of the L20 protein.

The use of chromatin immunoprecipitation to define PpsR binding activity in Rhodobacter sphaeroides 2.4.1.

The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA(-) mutant is unable to grow under photosynthetic conditions, when a ppsR mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an "extended" PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to R. sphaeroides. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation.

Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petIIABC gene cluster that encodes the three proteins, cytochrome c1, cytochrome b and a Rieske protein, that constitute a bc1 electron-transfer complex. RT-PCR and Northern blotting show that the petIIABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the c4 family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron-sulfur protein, suggesting that the six genes constitute an operon, termed the petII operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petI operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petI is transcribed mainly in cells grown in medium containing iron, whereas petII is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petI and petII operons. A model is presented in which petI is proposed to encode the bc1 complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petII is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional bc1 complexes.

Structural analysis of the HiPIP from the acidophilic bacteria: Acidithiobacillus ferrooxidans.

Hip is a high-potential iron-sulfur protein (HiPIP) isolated from the acidophilic bacterium, Acidithiobacillus ferrooxidans. In the present work, a structural model of Hip suggests that the role of proline residues is essential to stabilize the protein folding at very low pH. The presence of an unusual disulfide bridge in Hip is demonstrated using mass spectrometry and nuclear magnetic resonance. This disulfide bridge is necessary to anchor the N-terminal extremity of the protein, but is not involved in the acid stability of Hip. The structural parameters correlated with the pH dependence of Hip redox potential are also analysed on the basis of this model. Given that the same structural features can enhance acidic stability and lead to elevated redox potentials, modulation of the redox potentials of electron carriers may be necessary to achieve electron transfer at very low pH.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms.

The gene encoding a putative high-potential iron-sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron-sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a DeltatatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c1 from the second A. ferrooxidans cytochrome bc1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

The bc(1) complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction. Is there a second bc(1) complex?

Acidithiobacillus ferrooxidans is an acidophilic chemolithotrophic bacterium that can grow in the presence of either a weak reductant, Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). Here we first review the latest findings about the uphill electron transfer pathway established in iron-grown A. ferrooxidans, which has been found to involve a bc(1) complex. We then provide evidence that this bc(1) complex cannot function in the forward direction (exergonic reaction), even with an appropriate substrate. A search for the sequence of the three redox subunits of the A. ferrooxidans bc(1) complex (strain ATCC 19859) in the complete genome sequence of the A. ferrooxidans ATCC 23270 strain showed the existence of two different bc(1) complexes in A. ferrooxidans. Cytochrome b and Rieske protein sequence comparisons allowed us to point out some sequence particularities of these proteins in A. ferrooxidans. Lastly, we discuss the possible reasons for the existence of two different "classical" bc(1) complexes and put forward some suggestions as to what role these putative complexes may play in this acidophilic chemolithotrophic bacterium.

Characterization of the petI and res operons of Acidithiobacillus ferrooxidans.

DNA sequence analysis and bioinformatic interpretations have identified two adjacent clusters of genes potentially involved in the formation of a bc1 complex and in the maturation of a cytochrome c-type protein in two strains (ATCC 19859 and ATCC 33020) of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans). Reverse transcriptase-PCR experiments suggest that the two clusters are organized as operons, and +1 start sites of transcription for the operons have been determined by primer extension experiments. Potential promoters have been identified. The presence of these operons lends support to a recent model of reverse electron flow and is consistent with previous reports of phenotypic switching in this bacterium.