Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

Acyl-CoA thioesterases (ACOTs) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA-SH. In this study, we show that the expression profile of the ACOT isoforms changes remarkably during the differentiation of cultured rat brown adipocytes. Immunocytochemistry suggested that cytosolic ACOT1 was present in the preadipocytes, while mitochondrial ACOT2 was additionally expressed as the cells differentiated, concurrent with the accumulation of lipid droplets in the cytoplasm. Western blotting confirmed that, in contrast to ACOT1, the ACOT2 expression level was very low in the preadipocytes. However, after differentiation, the ACOT1 level fell to one-half of the baseline level and ACOT2 increased 18-fold. ACOT2 expression in the differentiated adipocytes was further enhanced by treatment with lipids or troglitazone. These changes in the ACOT2 expression level correlated well with changes in the expression of carnitine palmitoyltransferase 2, a mitochondrial β-oxidation enzyme. These results indicate that, in differentiating brown adipocytes, cytosolic ACOT1 becomes downregulated as the cellular use of acyl-CoA increases, while mitochondrial ACOT2 is upregulated as the β-oxidation capacity increases. ACOT isoform expression may be regulated during brown adipocyte differentiation to support the fat storage and combustion characteristics of this cell type.     

The role of the charged residues of the GP2 helical regions in Ebola entry

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.     

Ebola virus entry requires the host-programmed recognition of an intracellular receptor

Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appea...     

Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid‐enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA‐approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline‐treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.     

扎伊尔埃博拉病毒糖蛋白基因的克隆和表达

埃博拉病毒属丝状病毒科,能引发动物和人出血热症状,人感染后病死率高达90%以上,目前还没有有效预防和治疗的药物和疫苗。近年来,这种烈性传染病病毒传入我国的可能性不断加大,给我国公共卫生应急体系带来新的挑战。本研究针对埃博拉病毒的最主要结构蛋白——糖蛋白(GP),构建了重组原核表达载体pET28a(+)-GP1(33~313aa)、pET28a(+)-GP1(190~313aa)、pET28a(+)-GP2(502~632aa)、pET28a(+)-sGP,以及重组真核表达载体pcDNA3.1(+)-edited GP、pcDNA3.1(+)-GP1、pcDNA3.1(+)-GP。结果表明,GP1(33~313aa)、GP1(190~313aa)和sGP能在大肠埃希菌BL21(DE3)中以包涵体的形式表达,GP、GP1和GP2能在HEK293T细胞中表达,但均不能在BHK21细胞中表达。本研究为进一步探索埃博拉病毒GP的结构和功能及GP抗体制备奠定了基础。     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]     

Identification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein

The Ebola virus (EBOV) is a very contagious virus that is highly fatal in humans and animals. The largest epidemic was in West Africa in 2014, in which over 11,000 people died. However, to date, there are no licensed vaccines against it. Studies show that CD4⁺ and CD8⁺ T-cell responses, especially cytotoxic T-lymphocyte (CTL) responses, play key roles in protecting individuals from EBOV infection. Since HLA-restricted epitope vaccines are likely to be effective and safe immunization strategies for infectious diseases, the present study screened for CTL epitopes in the EBOV-nucleoprotein that are restricted by HLA-A11 (a common allele in Chinese people). Predictive computer analysis of the amino-acid sequence of EBOV-nucleoprotein identified ten putative HLA-A11-restricted epitopes. ELISPOT assay of immunized HLA-A11/DR1 transgenic mice showed that five (GR-9, VR-9, EK-9, PK-9, and RK-9) induced effective CTL responses. Additional epitope analyses will aid the design of epitope vaccines against EBOV.     

表达EB病毒主要膜蛋白的非复制型重组痘苗病毒毒力及体液免疫反应

利用表达EBV GP350/220的非复制型重组痘苗病毒VMA△CK及复制性重组痘苗病毒VMA,注射乳鼠脑内,观察毒力。同时,用VMA△CK及VMA免疫Bal/c小鼠,经ELISA测定其特异性抗体水平。免疫血清用抗体中和EBV感染Raji细胞抑制产生早期抗原（NEA）法测定其中和抗体滴度。将免疫血清与P3HR-1细胞共同孵育后,测上清中EBV滴度以观察抗体体制EBV从P3HR-1细胞中释放效应。结果发现,VMA△CK毒力明显低于VMA,其LD50为4.5PFU/0.02ml,而VMA△CK的LD50大于10^6PFU/0.02ml。VMA△CK与VAM免疫血清中抗GP350/220抗体水平无明显差异,而初兔后抗痘苗病毒抗体水平VMA免疫组较VMA△CK免疫组高5倍左右。VMA△CK免疫组血清中和抗体水平没有明显差异。VMA△CK免疫血清稀释度与P3HR-1细胞培养上清中EBV滴度有剂量依赖关系。     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p inhibitors can reduce the cytotoxicity of Ebola virus glycoprotein in vitro

Ebola virus(EBOV)causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91%in Zaire,the most lethal strain.Though the viral envelope glycoprotein(GP)mediates widespread inflammation and cellular damage,these changes have mainly focused on alterations at the protein level,the role of microRNAs(miRNAs)in the molecular pathogenesis underlying this lethal disease is not fully understood.Here,we report that the miRNAs hsa-miR-1246,hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells(HUVECs)following expression of EBOV GP.Among the proteins encoded by predicted targets of these miRNAs,the adhesion-related molecules tissue factor pathway inhibitor(TFPI),dystroglycan1(DAG1)and the caspase 8 and FADD-like apoptosis regulator(CFLAR)were significantly downregulated in EBOV GP-expressing HUVECs.Moreover,inhibition of hsa-miR-1246,hsa-miR-320a and hsa-miR-196b-5p,or overexpression of TFPI,DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP.Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.     

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC₅₀) value of each peptide was greater than 1000 μg mL⁻¹. The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL⁻¹ concentration showed a reduction of more than log₁₀ 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log₁₀ 4.8 to log₁₀ 2.5 and log₁₀ 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL⁻¹) to exert its anti-PPRV effect was much less than its CC₅₀ level (>1000 μg mL⁻¹). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.     

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.     

Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is broadly accepted that SARS-CoV-2 utilizes its spike protein to recognize the extracellular domain of angiotensin-converting enzyme 2 (ACE2) to enter cells for viral infection. However, other mechanisms of SARS-CoV-2 cell entry may occur. We show quantitatively that the SARS-CoV-2 spike protein also binds to the extracellular domain of broadly expressed integrin α5β1 with an affinity comparable to that of SARS-CoV-2 binding to ACE2. More importantly, we provide direct evidence that such binding promotes the internalization of SARS-CoV-2 into non-ACE2 cells in a manner critically dependent upon the activation of the integrin. Our data demonstrate an alternative pathway for the cell entry of SARS-CoV-2, suggesting that upon initial ACE2-mediated invasion of the virus in the respiratory system, which is known to trigger an immune response and secretion of cytokines to activate integrin, the integrin-mediated cell invasion of SARS-CoV-2 into the respiratory system and other organs becomes effective, thereby promoting further infection and progression of COVID-19.     

Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.     

Induction of humoral and cellular immune responses in mice by a recombinant fowlpox virus expressing the E2 protein of bovine viral diarrhea virus

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.