The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells.

The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells.

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism’s response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.

Liver Disease and Coronavirus Disease 2019: From Pathogenesis to Clinical Care.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus that emerged in late 2019, is posing an unprecedented challenge to global health. Coronavirus disease 2019 (COVID-19), the clinical disease caused by SARS-CoV-2, has a variable presentation ranging from asymptomatic infection to life-threatening acute respiratory distress syndrome and multiorgan failure. Liver involvement is common during COVID-19 and exhibits a spectrum of clinical manifestations from asymptomatic elevations of liver function tests to hepatic decompensation. The presence of abnormal liver tests has been associated with a more severe presentation of COVID-19 disease and overall mortality. Although SARS-CoV-2 RNA has been detected in the liver of patients with COVID-19, it remains unclear whether SARS-CoV-2 productively infects and replicates in liver cells and has a direct liver-pathogenic effect. The cause of liver injury in COVID-19 can be attributed to multiple factors, including virus-induced systemic inflammation, hypoxia, hepatic congestion, and drug-induced liver disease. Among patients with cirrhosis, COVID-19 has been associated with hepatic decompensation and liver-related mortality. Additionally, COVID-19's impact on health care resources can adversely affect delivery of care and outcomes of patients with chronic liver disease. Understanding the underlying mechanisms of liver injury during COVID-19 will be important in the management of patients with COVID-19, especially those with advanced liver disease. This review summarizes our current knowledge of SARS-CoV-2 virus-host interactions in the liver as well the clinical impact of liver disease in COVID-19.

Hepatitis C Virus and Hepatocellular Carcinoma: When the Host Loses Its Grip.

Chronic infection with hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC). Novel treatments with direct-acting antivirals achieve high rates of sustained virologic response; however, the HCC risk remains elevated in cured patients, especially those with advanced liver disease. Long-term HCV infection causes a persistent and accumulating damage of the liver due to a combination of direct and indirect pro-oncogenic mechanisms. This review describes the processes involved in virus-induced disease progression by viral proteins, derailed signaling, immunity, and persistent epigenetic deregulation, which may be instrumental to develop urgently needed prognostic biomarkers and as targets for novel chemopreventive therapies.

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

The interferon-induced transmembrane proteins 1-3 (IFITM1-3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1-3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein-protein interactions. Coexpression of IFITM1-3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

The Innate Antiviral Response in Animals: An Evolutionary Perspective from Flagellates to Humans.

Animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. Our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. However, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. In this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. We will then focus on some central conserved players of this response including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and cGAS-STING, attempting to put their evolution into perspective. To conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. These concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease.

Repertoire and Neutralizing Activity of Antibodies Against Hepatitis C Virus E2 Peptide in Patients With Spontaneous Resolution of Hepatitis C.

Neutralizing antibodies can prevent hepatitis C virus (HCV) infection, one of the leading causes of cirrhosis and liver cancer. Here, we characterized the immunoglobulin repertoire of memory B-cell antibodies against a linear epitope in the central front layer of the HCV envelope (E2; amino acids 483-499) in patients who were infected in a single-source outbreak. A reverse transcription polymerase chain reaction-based immunoglobulin gene cloning and recombinant expression approach was used to express monoclonal antibodies from HCV E2 peptide-binding immunoglobulin G-positive memory B cells. We identified highly mutated antibodies with a neutralizing effect in vitro against different genotype isolates sharing similar gene features. Our data confirm the importance of VH1-69 use for neutralizing activity. The data offer a promising basis for vaccine research and the use of anti-E2 antibodies as a means of passive immunization.

Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection.

Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.

In vivo combination of human anti-envelope glycoprotein E2 and -Claudin-1 monoclonal antibodies for prevention of hepatitis C virus infection.

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

Hepatitis C Virus (HCV)-Apolipoprotein Interactions and Immune Evasion and Their Impact on HCV Vaccine Design.

With more than 71 million people chronically infected, hepatitis C virus (HCV) is one of the leading causes of liver disease and hepatocellular carcinoma. While efficient antiviral therapies have entered clinical standard of care, the development of a protective vaccine is still elusive. Recent studies have shown that the HCV life cycle is closely linked to lipid metabolism. HCV virions associate with hepatocyte-derived lipoproteins to form infectious hybrid particles that have been termed lipo-viro-particles. The close association with lipoproteins is not only critical for virus entry and assembly but also plays an important role during viral pathogenesis and for viral evasion from neutralizing antibodies. In this review, we summarize recent findings on the functional role of apolipoproteins for HCV entry and assembly. Furthermore, we highlight the impact of HCV-apolipoprotein interactions for evasion from neutralizing antibodies and discuss the consequences for antiviral therapy and vaccine design. Understanding these interactions offers novel strategies for the development of an urgently needed protective vaccine.

Mapping Determinants of Virus Neutralization and Viral Escape for Rational Design of a Hepatitis C Virus Vaccine.

Hepatitis C virus (HCV) continues to spread worldwide with an annual increase of 1.75 million new infections. The number of HCV cases in the U.S. is now greater than the number of HIV cases and is increasing in young adults because of the opioid epidemic sweeping the country. HCV-related liver disease is the leading indication of liver transplantation. An effective vaccine is of paramount importance to control and prevent HCV infection. While this vaccine will need to induce both cellular and humoral immunity, this review is focused on the required antibody responses. For highly variable viruses, such as HCV, isolation and characterization of monoclonal antibodies mediating broad virus neutralization are an important guide for vaccine design. The viral envelope glycoproteins, E1 and E2, are the main targets of these antibodies. Epitopes on the E2 protein have been studied more extensively than epitopes on E1, due to higher antibody targeting that reflects these epitopes having higher degrees of immunogenicity. E2 epitopes are overall organized in discrete clusters of overlapping epitopes that ranged from high conservation to high variability. Other epitopes on E1 and E1E2 also are targets of neutralizing antibodies. Taken together, these regions are important for vaccine design. Another element in vaccine design is based on information on how the virus escapes from broadly neutralizing antibodies. Escape mutations can occur within the epitopes that are involved in antibody binding and in regions that are not involved in their epitopes, but nonetheless reduce the efficiency of neutralizing antibodies. An understanding on the specificities of a protective B cell response, the molecular locations of these epitopes on E1, E2, and E1E2, and the mechanisms, which enable the virus to negatively modulate neutralizing antibody responses to these regions will provide the necessary guidance for vaccine design.

Host-targeting therapies for hepatitis C virus infection: current developments and future applications.

Chronic hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases and hepatocellular carcinoma (HCC) worldwide. In the past few years, anti-HCV therapies have undergone a revolution with the approval of multiple direct-acting antivirals (DAAs), which enable interferon-free treatments with considerable improvement of sustained virologic response in patients. Today, DAAs have become the standard of care for HCV therapy. However, several limitations remain, which include access to therapy, treatment failure in a subset of patients and persistent risk of HCC development following cure in patients with advanced fibrosis. By targeting conserved host proteins involved in the HCV life cycle, host-targeting agents (HTAs) offer opportunities for pan-genotypic antiviral approaches with a high barrier to drug resistance. Moreover, when applied in combination with DAAs, HTAs could improve the management of difficult-to-treat patients by acting through a complementary mechanism of action. In this review, we summarize the different HTAs evaluated in preclinical and clinical development and discuss their potential role for anti-HCV therapies.

Rhesus macaque IFITM3 gene polymorphisms and SIV infection.

Interferon-induced transmembrane proteins (IFITMs) have been recognized as important antiviral effectors of the innate immune system, both in cell culture and in infected humans. In particular, polymorphisms of the human IFITM3 gene have been shown to affect disease severity and progression in influenza A virus (FLUAV) and human immunodeficiency virus (HIV) infection, respectively. Rhesus macaques (Macaca mulatta) are commonly used to model human infections and the experimental inoculation of these animals with simian immunodeficiency virus (SIV) is one of the best models for HIV/AIDS in humans. However, information on the role of IFITM3 in SIV infection of rhesus macaques is currently lacking. We show that rhesus macaque (rh) IFITM3 inhibits SIV and FLUAV entry in cell culture, although with moderately reduced efficiency as compared to its human counterpart. We further report the identification of 16 polymorphisms in the rhIFITM3 gene, three of which were exonic and synonymous while the remainder was located in non-coding regions. Employing previously characterized samples from two cohorts of SIV-infected rhesus macaques, we investigated the relationship between these rhIFITM3 polymorphisms and both AIDS-free survival time and virus load. In cohort 1, several intronic polymorphisms were significantly associated with virus load or survival. However, an association with both parameters was not observed and significance was lost in most cases when animals were stratified for the presence of MHC allele Mamu-A1*001. Moreover, no significant genotype-phenotype associations were detected in cohort 2. These results suggest that, although IFITM3 can inhibit SIV infection in cell culture, genetic variation in rhIFITM3 might have only a minor impact on the course of SIV infection in experimentally infected animals.

Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins.

Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate specific, raising the question of which parameters impact sensitivity to IFITM. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact the IFITM sensitivity of viral entry. IMPORTANCE: The host cell-encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact the sensitivity of viral entry to IFITMs. These results show for the first time that determinants other than the viral envelope protein can impact sensitivity to IFITM and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins.

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells.

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

Interferon-Induced Transmembrane Protein-Mediated Inhibition of Host Cell Entry of Ebolaviruses.

Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo.

Analysis of Ebola Virus Entry Into Macrophages.

Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.

Comparative Analysis of Host Cell Entry of Ebola Virus From Sierra Leone, 2014, and Zaire, 1976.

The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors.