Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA, the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development, but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HTR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HTR and clinically available 5-HTR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover, phenoxybenzamine (PBZ), an FDA-approved 5-HTR antagonist, inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry, but also present a promising target for the invention of HCV entry inhibitor.

RNA binding protein 24 regulates the translation and replication of hepatitis C virus

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5'-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5'-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5'- and 3'-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.

Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway / 中国病毒学·英文版

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle.In the present study using a Huh7 cell line Con1 with an HCV replicon,we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling.Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells.It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site.Consistently,a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays.Thus,the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication.In addition,cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine,an inhibitor of CDKs had a similar effect to that of U0126.Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels.Further,the replication of HCV replicon in Conl cells was inhibited by IFN-α.The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs.It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

The Protamine-like DNA-binding Protein P6.9 Epigenetically Up-regulates Autographa californica Multiple Nucleopolyhedrovirus Gene Transcription in the Late Infection Phase / 中国病毒学·英文版

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1].It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes P6.9,a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29].Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly,while it has no influence on viral genome replication[31].In the present study,the role of P6.9 in viral gene transcription regulation is characterized.In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription,P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection (hpi),whereas it is non-essential for the basal level of viral gene transcription.Fluorescence microscopy reveals the P6.9's co-localization with DNA is temporally and spatially synchronized with P6.9's impact on viral gene transcription,indicating the P6.9-DNA association contributes to transcription regulation.Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase Ⅱ in the same transcriptionally active chromatin fraction at 24 hpi,which may probably contribute to viral gene transcription up-regulation in the late infection phase.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication / 中国病毒学·英文版

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV)usually result in altered hepatitis B surface antigen(HBsAg)secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs)synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication / 中国病毒学·英文版

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPv) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Tbr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro / 中国病毒学·英文版

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.

The ORF 113 of Heliocoverpa armigera Single Nucleopolyhedrovirus Encodes a Functional Fibroblast Growth Factor / 中国病毒学·英文版

Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group Ⅱ NPV. The HearNPV vfgftranscripts were detected from 18 to 96 h post-infection (hpi) of Hz-AMI cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM 1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.

Expression of recombinant baculovirus carrying Schistosoma japonicum 26 ku GST in mammalian cells / 华中科技大学学报（医学）（英德文版）

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 x 10(8). Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells / 华中科技大学学报（医学）（英德文版）

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.