[Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein].

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-ß1 (TGF-ß1) or co-treated with TGF-ß1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-ß1 and 5 µmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-ß1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-ß1 single treatment group (P < 0.05). CONCLUSION: A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.

Proteomic screening identifies RPLp2 as a specific regulator for the translation of coronavirus.

Viral mRNA of coronavirus translates in an eIF4E-dependent manner, and the phosphorylation of eIF4E can modulate this process, but the role of p-eIF4E in coronavirus infection is not yet entirely evident. p-eIF4E favors the translation of selected mRNAs, specifically the mRNAs that encode proteins associated with cell proliferation, inflammation, the extracellular matrix, and tumor formation and metastasis. In the present work, two rounds of TMT relative quantitative proteomics were used to screen 77 cellular factors that are upregulated upon infection by coronavirus PEDV and are potentially susceptible to a high level of p-eIF4E. PEDV infection increased the translation level of ribosomal protein lateral stalk subunit RPLp2 (but not subunit RPLp0/1) in a p-eIF4E-dependent manner. The bicistronic dual-reporter assay and polysome profile showed that RPLp2 is essential for translating the viral mRNA of PEDV. RNA binding protein and immunoprecipitation assay showed that RPLp2 interacted with PEDV 5'UTR via association with eIF4E. Moreover, the cap pull-down assay showed that the viral nucleocapsid protein is recruited in m7GTP-precipitated complexes with the assistance of RPLp2. The heterogeneous ribosomes, which are different in composition, regulate the selective translation of specific mRNAs. Our study proves that viral mRNA and protein utilize translation factors and heterogeneous ribosomes for preferential translation initiation. This previously uncharacterized process may be involved in the selective translation of coronavirus.

Selective regulation in ribosome biogenesis and protein production for efficient viral translation.

As intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. Viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. Recently reported studies have demonstrated that ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs) act as multifaceted regulators in selective translation of viral transcripts. Here we summarize the recent literature on RBFs and RPs and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. The advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents.

The Natural Compound Homoharringtonine Presents Broad Antiviral Activity In Vitro and In Vivo.

To complement traditional antivirals, natural compounds that act via host targets and present high barriers to resistance are of increasing interest. In the work reported here, we detected that homoharringtonine (HHT) presents effective antiviral activity. HHT completely inhibited infections of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), and porcine epidemic diarrhea virus (PEDV) at concentrations of 50, 100, and 500 nM in cell cultures, respectively. Treatment with HHT at doses of 0.05 or 0.2 mg/kg significantly reduced viral load and relieved severe symptoms in PEDV- or NDV-infected animals. HHT treatment, however, moderately inhibited avian influenza virus (AIV) infection, suggesting its potent antiviral action is restricted to a number of classes of RNA viruses. In this study, we also observed that HHT actively inhibited herpes simplex virus type 1 (HSV-1) replication with a 50% inhibitory concentration (IC50) of 139 nM; the treatment with HHT at 1000 nM led to reductions of three orders of magnitude. Moreover, HHT antagonized the phosphorylation level of endogenous and exogenous eukaryotic initiation factor 4E (p-eIF4E), which might regulate the selective translation of specific messenger RNA (mRNA). HHT provides a starting point for further progress toward the clinical development of broad-spectrum antivirals.

Cytoplasmic Translocation of Nucleolar Protein NOP53 Promotes Viral Replication by Suppressing Host Defense.

NOP53 is a tumor suppressor protein located in the nucleolus and is translocated to the cytoplasm during infection by vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1), as shown in our previous study. Cytoplasmic NOP53 interacts with the retinoic acid-inducible gene I (RIG-I) to remove its K63-linked ubiquitination, leading to attenuation of type I interferon IFN-β. In the present study, we found no obvious translocation of NOP53 in infection by a mutant virus lacking ICP4 (HSV-1/d120, replication inadequate). Blocking cytoplasmic translocation of NOP53 by the deletion of its nuclear export sequence (NES) abrogated its ability to support viral replication. These results demonstrated that NOP53 redistribution is related to viral replication. It is interesting that treatment with poly (I:C) or RIG-I-N (a constitutively-active variant) directly induced NOP53 cytoplasmic translocation. To better assess the function of cytoplasmic NOP53 in viral replication, the NOP53-derived protein N3-T, which contains a human immunodeficiency virus (HIV)-derived cell-penetrating Tat peptide at the C-terminal region of N3 (residues 330⁻432), was constructed and expressed. The recombinant N3-T protein formed trimers, attenuated the expression of IFN-β and IFN-stimulated genes, as well as decreased the phosphorylation level of interferon regulatory factor 3 (IRF3). Furthermore, N3-T promoted the efficient replication of enveloped and non-enveloped DNA and RNA viruses belonging to 5 families. Our findings expand the understanding of the mechanism by which viruses utilize the nucleolar protein NOP53 for optimal viral replication.

Multifunctional viral protein γ34.5 manipulates nucleolar protein NOP53 for optimal viral replication of HSV-1.

To ensure efficient virus replication, herpes simplex virus type 1 (HSV-1) encodes several viral proteins to counter host defense response upon infection. Among these proteins, the multifunctional viral protein γ34.5 crucially interferes with or disrupts several antiviral pathways at multiple levels. The current study shows that γ34.5 utilizes nucleolar protein NOP53 to facilitate the dephosphorylation of eukaryotic initiation factor eIF2α for efficient viral translation. Our study shows that: (1) ectopic expression of NOP53 greatly increases the intracellular and extracellular viral yields of HSV-1 (wild strain F) in type I interferon-deficient Vero cells, and more subtly promotes viral replication of γ34.5 deletion mutant virus HSV-1/Δγ34.5. (2) NOP53 is migrated from nuclei in HSV-1/F infected cells, but is redistributed incompletely after infection by either HSV-1/Δγ34.5 or ICP4 deletion mutant virus HSV-1/d120 (replication inadequate). Ectopic expression of γ34.5, consequently, induces cytoplasmic translocation of NOP53 in response to HSV-1/Δγ34.5 infection. (3) Increase of NOP53, in two forms of transient transfection and in vitro expression, attenuates the phosphorylation level of eIF2α in HSV-1/F infected cells, but fails to affect eIF2α phosphorylation induced by HSV-1/Δγ34.5 infection. (4) Knockdown of NOP53, which impairs the specific interaction between γ34.5 and protein phosphatase PP1α, disrupts the ability of γ34.5 to maintain HSV-1 virulence. (5) NOP53 knockdown also significantly reduces tissue damage and decreases viral yield in livers of HSV-1 infected mice. Our findings expand the understanding of the underlying mechanism by which viral protein γ34.5 induces NOP53 redistribution; cytoplasmic NOP53 facilitates γ34.5 recruitment of PP1α to dephosphorylate eIF2α, for optimal viral replication. This paper also demonstrates that blocking the specific interaction between γ34.5 and PP1α would be a useful approach for the development of antiviral agents.

Cellular protein GLTSCR2: A valuable target for the development of broad-spectrum antivirals.

Viral infection induces translocation of the nucleolar protein GLTSCR2 from the nucleus to the cytoplasm, resulting in attenuation of the type I interferon IFN-ß. Addressing the role of GLTSCR2 in viral replication, we detect that knocking down GLTSCR2 by shRNAs results in significant suppression of viral replication in mammalian and chicken cells. Injection of chicken embryo with the GLTSCR2-specific shRNA-1370 simultaneously or 24 h prior to infection with Newcastle disease virus (NDV) substantially reduces viral replication in chicken embryo fibroblasts. Injection of shRNA-1370 into chicken embryo also reduces the replication of avian influenza virus (AIV). In contrast, GLTSCR2-derived protein G4-T, forming α-helical dimers, increases replication of seven various DNA and RNA viruses in cells. Our studies reveal that alteration of the function of cellular GLTSCR2 plays a role in supporting viral replication. GLTSCR2 should be seriously considered as a therapeutic target for developing broad spectrum antiviral agents to effectively control viral infection.

Expression of recombinant herpes simplex virus type 2 glycoprotein D by high-density cell culture of Spodoptera frugiperda.

Herpes simplex virus type 2 (HSV-2) is the major cause of genital herpes in humans. The glycoprotein D of HSV-2 (gD2) is a promising subunit vaccine candidate for the treatment of genital herpes. The aim of the present study was to express a biologically active recombinant gD2 in eukaryotic baculovirus system in quantities sufficient for further studies. Human cDNA encoding a gD2 protein with 393 amino acids was subcloned into the pFastBac HTb vector and the recombinant protein was expressed in Spodoptera frugiperda (Sf9) cells by high-density cell culture. In a stirred bioreactor, the key limiting factors including glucose concentration, glutamine concentration and dissolved oxygen (DO) were optimized for high-density cell growth. The Sf9 cell density could reach 9.6×106 cells/mL and the yield of recombinant gD2 protein was up to 192 mg/L in cell culture under the optimal conditions of 15 mM glucose, 0.4 g/L glutamine and 40% DO. Production of significant amounts of pure, full-length gD2 opened up the possibility to investigate novel functions of gD2. Moreover, the purified recombinant gD2 protein revealed a partial prophylactic immune function in genital herpes of guinea pigs infected with HSV-2.

The distinct anchoring mechanism of FtsY from different microbes.

The SRP receptor FtsY, which is involved in targeting and translocating membrane protein, is generally composed of the N-terminal domain and the NG domain. Although FtsY was highly homologous in the composition of amino acids and functions among microbes, the different mechanism in the location of FtsYs from different bacteria such as S. coelicolor and E. coli were discovered in this study by laser scanning confocal microscope (LSCM) in vivo and molecular techniques in vitro. The results revealed that the N-terminal domain of S. coelicolor FtsY was indispensable for FtsY's anchoring membrane, and while the A domain of E. coli FtsY was dispensable. Moreover, the A domain of E. coli FtsY might promote itself to bind the membrane depending on the location images and Western blotting.

FtsY affects sporulation and antibiotic production by whiH in Streptomyces coelicolor.

FtsY, the Signal Recognition Particle (SRP) receptor in bacteria, is known to facilitate the cotranslational protein targeting by recruiting SRP-protein complex to secYEG. We show in this work that deletion of the ftsY gene in Streptomyces coelicolor would also lead to complete blockage of sporulation process and reduced production of antibiotic actinorhodin. These defects cannot be complemented by only the NG domain of FtsY, while full-length ftsY was able to restore spore generation and increase production of actinorhodin in ftsY-disrupted strains. Further transcriptional analysis on sporulation controlling genes, i.e., whiG, whiB, whiH, and prox, indicated that the regulation of sporulation by ftsY is likely to take effect through whiH.

Functional characterization of Streptomyces coelicolor FtsY.

This study indicated that the N-terminus was dispensable for FtsY GTPase activity, and that the N-domain plays an essential role in the GTPase activity of the NG domain. In addition, the S.scoelicolor FtsY was able to restore function in an E. coli mutant. However, its NG domain was unable to play any roles.

Analysis of the GTPase activity and active sites of the NG domains of FtsY and Ffh from Streptomyces coelicolor.

Fifty-four homolog (Ffh) and FtsY are the central components of the signal recognition particle secretory pathway of bacteria. In this study, the core domain and active sites of FtsY and Ffh from Streptomyces coelicolor, which are responsible for guanosine triphosphate (GTP) hydrolysis, were identified using site-directed mutagenesis. Mutations were introduced to the conserved GXXGXGK loop of the putative GTP binding site. Mutation of the Lys residue to Gly in both FtsY and Ffh NG domains significantly decreased the GTPase activity and GTP binding affinity. Furthermore, a structural model of the ternary complex of FtsY/Ffh NG domains and the non-hydrolyzable GTP analog guanylyl 5'-(beta,gamma-methylenediphosphonate) also revealed that each Lys residue in GXXGXGK of FtsY and Ffh provides the predicted hydrogen bond required for GTP binding. However, in FtsY not in Ffh, mutation of the first Gly residue in the GXXGXGK loop disrupted the GTPase activity. In addition, protease-digesting test demonstrated that NG protein with the mutation of Lys residue was decomposed more easily. Western blot analysis suggested that in Streptomyces coelicolor, FtsY is present in the membrane fraction and Ffh in the cytosol fraction during the mid-log phase of growth. These results indicated that Lys residue in the putative GTP binding loop was the crucial residue for the GTPase activity of NG domain.

Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor.

Non-optimal (rare) codons have been suggested to reduce translation rate and facilitate secretion in Escherichia coli. In this study, the complete genome analysis of non-optimal codon usage in secretory signal sequences and non-secretory sequences of Streptomyces coelicolor was performed. The result showed that there was a higher proportion of non-optimal codons in secretory signal sequences than in non-secretory sequences. The increased tendency was more obvious when tested with the experimental data of secretory proteins from proteomics analysis. Some non-optimal codons for Arg (AGA, CGU and CGA), Ile (AUA) and Lys (AAA) were significantly over presented in the secretary signal sequences. It may reveal that a balanced non-optimal codon usage was necessary for protein secretion and expression in Streptomyces.

[Research progress of targeting and translocation of proteins mediated by signal recognition particle in prokaryote].

How proteins are targeted and translocated mediated by signal recognition particle (SRP) in eukaryotic cell is very clear and uniform. In contrast, SRP systems are different in various kinds of prokaryotic cells, So it is difficult to identify. Nowadays, the studies of prokaryotic SRP system focus on the structure and function of Ffh, FtsY, 4.5S RNA, and GTP as a regulating molecular. Here, a description was given on research progress of constitutes, structures and functions of bacterial SRP complex proteins. The research status of streptomyces SRP pathway was also reviewed, and this study in streptomyces will be helpful to explain the molecular mechanism of prokaryotic SRP system.