Type I interferon signaling induces a delayed antiproliferative response in respiratory epithelial cells during SARS-CoV-2 infection.

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

A specific EMC subunit supports Dengue virus infection by promoting virus membrane fusion essential for cytosolic genome delivery.

Dengue virus (DENV) represents the most common human arboviral infection, yet its cellular entry mechanism remains unclear. The multi-subunit endoplasmic reticulum membrane complex (EMC) supports DENV infection, in part, by assisting the biosynthesis of viral proteins critical for downstream replication steps. Intriguingly, the EMC has also been shown to act at an earlier step prior to viral protein biogenesis, although this event is not well-defined. Here we demonstrate that the EMC subunit EMC4 promotes fusion of the DENV and endosomal membranes during entry, enabling delivery of the viral genome into the cytosol which is then targeted to the ER for viral protein biosynthesis. We also found that EMC4 mediates ER-to-endosome transfer of phosphatidylserine, a phospholipid whose presence in the endosome facilitates DENV-endosomal membrane fusion. These findings clarify the EMC-dependent DENV early entry step, suggesting a mechanism by which an ER-localized host factor can regulate viral fusion at the endosome.

Identification of cell type specific ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

What faculty write versus what students see? Perspectives on multiple-choice questions using Bloom's taxonomy.

BACKGROUND: Using revised Bloom's taxonomy, some medical educators assume they can write multiple choice questions (MCQs) that specifically assess higher (analyze, apply) versus lower-order (recall) learning. The purpose of this study was to determine whether three key stakeholder groups (students, faculty, and education assessment experts) assign MCQs the same higher- or lower-order level. METHODS: In Phase 1, stakeholders' groups assigned 90 MCQs to Bloom's levels. In Phase 2, faculty wrote 25 MCQs specifically intended as higher- or lower-order. Then, 10 students assigned these questions to Bloom's levels. RESULTS: In Phase 1, there was low interrater reliability within the student group (Krippendorf's alpha = 0.37), the faculty group (alpha = 0.37), and among three groups (alpha = 0.34) when assigning questions as higher- or lower-order. The assessment team alone had high interrater reliability (alpha = 0.90). In Phase 2, 63% of students agreed with the faculty as to whether the MCQs were higher- or lower-order. There was low agreement between paired faculty and student ratings (Cohen's Kappa range .098-.448, mean .256). DISCUSSION: For many questions, faculty and students did not agree whether the questions were lower- or higher-order. While faculty may try to target specific levels of knowledge or clinical reasoning, students may approach the questions differently than intended.

Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle.

The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.

Functional Analysis of the Dengue Virus Genome Using an Insertional Mutagenesis Screen.

In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genomewide transposon mutagenesis screen with next-generation sequencing to determine sites in the viral genome that tolerate 15-nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection and found 6 of them were capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.IMPORTANCE Dengue virus is a mosquito-borne virus distributed in tropical and subtropical regions globally that can result in hospitalization and even death in some cases. Although a vaccine exists, its limitations and a lack of approved antiviral treatments highlight our limited understanding of dengue virus pathogenesis and host immunity. The functions of many viral proteins are poorly understood. We used a previously published approach using transposon mutagenesis to develop tools to study these proteins' functions by adding insertions randomly throughout the viral genomes. These genomes were transferred into cells, and infectious progeny were recovered to determine sites that tolerated insertions, as only the genomes that tolerated insertions would be able to propagate. Using these results, we created viruses with epitope tags, one in the viral structural protein Capsid and one in the viral nonstructural protein NS4B. Further investigation of these mutants may elucidate the roles of Capsid and NS4B during dengue virus infections.

Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging.

BACKGROUND & AIMS: Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation. METHODS: Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time. RESULTS: We found that HCV replication organelles are continuously generated de novo at spatially distinct sites from preformed ones. This process is accompanied by accumulated intracellular membrane alteration, increased cholesterol delivery, NS5A phosphorylation, and positive-strand RNA content, and by eventual association with HCV core protein around lipid droplets. Generation of spatially segregated foci requires viral NS5A and the host factors phosphatidylinositol 4-kinase and oxysterol-binding protein, while association of foci with lipid droplets requires cholesterol. CONCLUSIONS: Our results reveal that HCV replication organelles are not static structures, but instead are continuously generated and dynamically change in composition and possibly also in function. LAY SUMMARY: Hepatitis C virus replication membrane structures are continuously generated at spatially distinct sites. New replication organelles are different in composition, and possibly also in function, compared to old replication organelles.

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos.

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.

Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.

BACKGROUND & AIMS: Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication. In addition, although the HCV replication compartment, known as the membranous web, is believed to be cholesterol enriched, the mechanisms by which this occurs have not been elucidated. We aimed to identify and characterize a PI 4-kinase effector in HCV replication. METHODS: We used a combination of microscopic and biochemical methods to study HCV replication, web morphology, the distribution of intracellular protein and PI(4)P, along with cholesterol trafficking in HCV-infected cells. PI 4-kinase and oxysterol-binding protein (OSBP) were inhibited using RNA interference or small molecules in cells expressing a full-length genotype 1b replicon or infected with the JFH-1 strain of HCV. RESULTS: OSBP was required for HCV replication and membranous web integrity. OSBP was recruited to membranous webs in a PI 4-kinase-dependent manner, and both these factors were found to regulate cholesterol trafficking to the web. We also found OSBP to be required for poliovirus infection but dispensable for dengue virus. CONCLUSIONS: OSBP is a PI 4-kinase effector in HCV infection, and contributes to the integrity and cholesterol enrichment of the membranous web. OSBP might also be a PI 4-kinase effector in poliovirus infection and could be involved in replication of other viruses that require PI 4-kinases.

Rab18 binds to hepatitis C virus NS5A and promotes interaction between sites of viral replication and lipid droplets.

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.

Self-Assembled STING-Activating Coordination Nanoparticles for Cancer Immunotherapy and Vaccine Applications.

The cGAS-STING pathway plays a crucial role in innate immune activation against cancer and infections, and STING agonists based on cyclic dinucleotides (CDN) have garnered attention for their potential use in cancer immunotherapy and vaccines. However, the limited drug-like properties of CDN necessitate an efficient delivery system to the immune system. To address these challenges, we developed an immunostimulatory delivery system for STING agonists. Here, we have examined aqueous coordination interactions between CDN and metal ions and report that CDN mixed with Zn2+ and Mn2+ formed distinctive crystal structures. Further pharmaceutical engineering led to the development of a functional coordination nanoparticle, termed the Zinc-Mn-CDN Particle (ZMCP), produced by a simple aqueous one-pot synthesis. Local or systemic administration of ZMCP exerted robust antitumor efficacy in mice. Importantly, recombinant protein antigens from SARS-CoV-2 can be simply loaded during the aqueous one-pot synthesis. The resulting ZMCP antigens elicited strong cellular and humoral immune responses that neutralized SARS-CoV-2, highlighting ZMCP as a self-adjuvant vaccine platform against COVID-19 and other infectious pathogens. Overall, this work establishes a paradigm for developing translational coordination nanomedicine based on drug-metal ion coordination and broadens the applicability of coordination medicine for the delivery of proteins and other biologics.

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

BACKGROUND & AIMS: Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. METHODS: We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. RESULTS: CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. CONCLUSIONS: HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection.

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

STING agonist-loaded mesoporous manganese-silica nanoparticles for vaccine applications.

Cyclic dinucleotides (CDNs), as one type of Stimulator of Interferon Genes (STING) pathway agonist, have shown promising results for eliciting immune responses against cancer and viral infection. However, the suboptimal drug-like properties of conventional CDNs, including their short in vivo half-life and poor cellular permeability, compromise their therapeutic efficacy. In this study, we have developed a manganese-silica nanoplatform (MnOx@HMSN) that enhances the adjuvant effects of CDN by achieving synergy with Mn2+ for vaccination against cancer and SARS-CoV-2. MnOx@HMSN with large mesopores were efficiently co-loaded with CDN and peptide/protein antigens. MnOx@HMSN(CDA) amplified the activation of the STING pathway and enhanced the production of type-I interferons and other proinflammatory cytokines from dendritic cells. MnOx@HMSN(CDA) carrying cancer neoantigens elicited robust antitumor T-cell immunity with therapeutic efficacy in two different murine tumor models. Furthermore, MnOx@HMSN(CDA) loaded with SARS-CoV-2 antigen achieved strong and durable (up to one year) humoral immune responses with neutralizing capability. These results demonstrate that MnOx@HMSN(CDA) is a versatile nanoplatform for vaccine applications.

Genetic and functional studies of phosphatidyl-inositol 4-kinase type IIIα.

Phosphatidylinositol 4-kinase type IIIa (PI4KIIIα) is one of four mammalian PI 4-kinases that catalyzes the first committed step in polyphosphoinositide synthesis. PI4KIIIα has been linked to regulation of ER exit sites and to the synthesis of plasma membrane phosphoinositides and recent studies have also revealed its importance in replication of the Hepatitis C virus in liver. Two isoforms of the mammalian PI4KIIIα have been described and annotated in GenBank: a larger, ~230kDa (isoform 2) and a shorter splice variant containing only the ~97kDa C-terminus that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of ~7.5kb with the exception of the proerythroleukemia line K562, which contained significantly higher level of the 7.5kb transcript along with smaller ones of 2.4, 3.5 and 4.2kb size. Bioinformatic analysis also confirmed the high copy number of PI4KIIIα transcript in K562 cells along with several genes located in the same region in Chr22, including two pseudogenes that cover most exons coding for isoform 1, consistent with chromosome amplification. A panel of polyclonal antibodies raised against peptides within the C-terminal half of PI4KIIIα failed to detect the shorter isoform 1 either in COS-7 cells or K562 cells. Moreover, expression of a cDNA encoding isoform 1 yielded a protein of ~97kDa that showed no catalytic activity and failed to rescue hepatitis C virus replication. These data draw attention to PI4KIIIα as one of the genes found in Chr22q11, a region affected by chromosomal instability, but do not substantiate the existence of a functionally relevant short form of PI4KIIIα.

A functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis C virus.

BACKGROUND & AIMS: The precise mechanisms by which IFN exerts its antiviral effect against HCV have not yet been elucidated. We sought to identify host genes that mediate the antiviral effect of IFN-α by conducting a whole-genome siRNA library screen. METHODS: High throughput screening was performed using an HCV genotype 1b replicon, pRep-Feo. Those pools with replicate robust Z scores ≥2.0 entered secondary validation in full-length OR6 replicon cells. Huh7.5.1 cells infected with JFH1 were then used to validate the rescue efficacy of selected genes for HCV replication under IFN-α treatment. RESULTS: We identified and confirmed 93 human genes involved in the IFN-α anti-HCV effect using a whole-genome siRNA library. Gene ontology analysis revealed that mRNA processing (23 genes, p=2.756e-22), translation initiation (nine genes, p=2.42e-6), and IFN signaling (five genes, p=1.00e-3) were the most enriched functional groups. Nine genes were components of U4/U6.U5 tri-snRNP. We confirmed that silencing squamous cell carcinoma antigen recognized by T cells (SART1), a specific factor of tri-snRNP, abrogates IFN-α's suppressive effects against HCV in both replicon cells and JFH1 infectious cells. We further found that SART1 was not IFN-α inducible, and its anti-HCV effector in the JFH1 infectious model was through regulation of interferon stimulated genes (ISGs) with or without IFN-α. CONCLUSIONS: We identified 93 genes that mediate the anti-HCV effect of IFN-α through genome-wide siRNA screening; 23 and nine genes were involved in mRNA processing and translation initiation, respectively. These findings reveal an unexpected role for mRNA processing in generation of the antiviral state, and suggest a new avenue for therapeutic development in HCV.

Hepatitis B virus core promoter mutations contribute to hepatocarcinogenesis by deregulating SKP2 and its target, p21.

BACKGROUND & AIMS: Clinical studies have associated hepatitis B virus core promoter (CP) mutations with an increased risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been implicated in hepatocarcinogenesis. The cyclin kinase inhibitor p21WAF1/CIP1 is an important regulator of cell cycle progression and proliferation. We determined whether HBx mutants that result from mutations in the CP deregulate p21 and these processes. METHODS: We constructed a series of HBx mutants with changes in the CP region that correspond to A1762T/G1764A (TA), T1753A, T1768A, or a combination of these (combo) and expressed them, along with wild-type HBx under control of its endogenous promoter, in primary human hepatocytes (PHHs) and HepG2 cells. We then analyzed the effects of CP mutations on expression and degradation of p21 and the effects on cell cycle progression and proliferation. RESULTS: The combo mutant decreased levels of p21 and increased cyclin E expression in PHHs and HepG2 cells. The combo mutant, but not HBx with single or double CP mutations, accelerated p21 degradation in HepG2 cells. The combo mutant increased expression of S-phase kinase-associated protein 2 (SKP2) in PHHs and Huh7 cells. Silencing of SKP2 abrogated the effects of CP mutations on p21 expression. The kinetics of p21 expression correlated with changes in cell cycle distribution. The combo mutant accelerated cell cycle progression; p21 overexpression restored G1 arrest. CONCLUSIONS: HBx mutants with changes that correspond to a combination of CP mutations up-regulate SKP2, which then down-regulates p21 via ubiquitin-mediated proteasomal degradation. CP mutations might increase the risk of hepatocellular carcinoma via this pathway.

SARS-CoV-2 triggers Golgi fragmentation via down-regulation of GRASP55 to facilitate viral trafficking.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped RNA virus. Despite the high economic and life losses caused by SARS-CoV-2, the detailed viral cycle, especially how it assembles and traffics in the secretory pathway, remains largely unknown. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. Disrupting Golgi function with small molecules strongly inhibits viral infection. Furthermore, expression of several SARS-CoV-2 proteins individually is sufficient to trigger Golgi fragmentation. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 expression, while expression of GRASP55 or knockdown of TGN46 reduces the infection rate of both USA-WA1 and Delta variants of SARS-CoV-2. Our study reveals that SARS-CoV-2 modulates Golgi structure and function via altering GRASP55 and TGN46 expression to facilitate viral trafficking, indicating the Golgi as a novel therapeutic target to block SARS-CoV-2 infection.