Nuclear beta-arrestin1 functions as a scaffold for the dephosphorylation of STAT1 and moderates the antiviral activity of IFN-gamma.

Signal transducers and activators of transcription 1 (STAT1) is activated by tyrosine phosphorylation upon interferon-gamma (IFN-gamma) stimulation. Phosphorylated STAT1 translocates into nucleus to initiate the transcription of IFN-gamma target genes that are important in mediating antiviral, antiproliferative, and immune response. The inactivation of STAT1 is mainly accomplished via tyrosine dephosphorylation by the nuclear isoform of T cell protein tyrosine phosphatase (TC45) in nucleus. Here we show that beta-arrestin1 directly interacts with STAT1 in nucleus after IFN-gamma treatment and accelerates STAT1 tyrosine dephosphorylation by recruiting TC45. Consequently, beta-arrestin1 negatively regulates STAT1 transcription activity as well as the IFN-gamma-induced gene transcription. Application of beta-arrestin1 siRNA significantly enhances IFN-gamma-induced antiviral response in vesicular stomatitis virus (VSV)-infected cells. Our results reveal that nuclear beta-arrestin1, acting as a scaffold for the dephosphorylation of STAT1, is an essential negative regulator of IFN-gamma signaling and participates in the IFN-gamma-induced cellular antiviral response.

Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes.

Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.

Hepatitis C virus genotype diversity in Shanghai, China.

We studied 67 hepatitis C virus (HCV) isolates from 64 hospitalized patients in Shanghai, China. Genotype 1 was prevalent, and genotypes 2, 3, 6 were found for the first time in Shanghai. A rare mixed infection with three subtypes (1a, 1b, 2a) was found. The complete genome sequence of a subtype 3b isolate was determined and analyzed.

Kinetic simulation study of femtosecond laser processing of graphene oxide: first-principles.

CONTEXT: Organic-inorganic nanoparticles have received extensive attention in various fields due to their unique physicochemical properties and biological activities. Among these nanoparticles, graphene oxide (GO) has emerged as a promising material, and thus, its application in biomedical fields is of great interest. Coating graphene oxide on the surface of implants can enhance its properties such as antibacterial and cell proliferation promotion, but the osteogenic properties of graphene oxide coating need further improvement, and the chance of acute inflammation triggered by local reactive oxygen species accumulation needs to be reduced. High-precision modulation of graphene oxide surface micro/nanomorphology and chemical composition can be achieved using femtosecond laser processing technology to improve its performance while also reducing the oxygen content of the graphene oxide surface to some extent. In this paper, the properties of graphene oxide were investigated by kinetic simulations based on the first-principle. The results show that the band gap of graphene oxide changes from 0.386 to 0.021 eV; the work function changes from 4.882 to 4.64 eV; the size and number of peaks in the radial distribution function decreases; and the intensity of the scatter X-ray peak becomes smaller under the action of femtosecond laser, indicating that the oxygen-containing functional groups on the surface of graphene oxide are disrupted, which provides a basis for its potential application in the medical field. METHODS: To investigate the properties of graphene oxide, SEM, XPS, Raman, and FTIR characterizations were first used to determine the oxygen-containing functional group species on the surface of graphene oxide. The structural model of graphene oxide was then modeled for density flooding theory (DFT) simulations using Biovia Materials Studio software, which was implemented in the CASTEP code. Our DFT calculations were performed using the generalized gradient approximation (GGA) as parameterized by the Perdew-Burke Ernzerhof (PBE) exchange-correlation functional. Additionally, we employed the norm-conserving pseudopotential to treat core electrons.

Inhibition of hepatitis B virus replication by MyD88 involves accelerated degradation of pregenomic RNA and nuclear retention of pre-S/S RNAs.

Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-alpha), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-alpha-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5'-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88.

Identification of recombination between subgenotypes IA and IB of hepatitis A virus.

Co-circulation of subgenotypes IA and IB of hepatitis A virus (HAV) has been reported in South Africa, South America, Europe, and the United States. In this study, phylogenetic and recombination analyses were performed for the first time on 31 complete HAV genomes from infected humans and simians. Three potentially significant intra-genotypic recombination events (I-III) were identified by recombination detection analysis. Recombination events I and II occurred between the lineages represented, respectively, by the Japanese isolate AH2 (AB020565, subgenotype IA) and the North African isolate MBB (M20273, subgenotype IB), giving rise to the recombinant Uruguayan isolate HAV5 (EU131373). Recombination event III occurred between the lineages represented, respectively, by the North African isolate MBB (M20273, subgenotype IB) and the German isolate GBM (X75215, subgenotype IA), resulting in the Italian isolate FG (X83302). The findings demonstrate that humans can be co-infected with different HAV subgenotypes and provide valuable hints for future research on HAV diversity.

[Phacoemulsification cataract surgery with different cumulative energy composite parameters in patients with type 2 diabetes mellitus:therapeutic effect and complications].

OBJECTIVE: To evaluate the effect of different cumulative energy composite parameters on the outcomes of phacoemulsification cataract surgery in patients with type 2 diabetes mellitus. METHODS: A total of 252 patients with cataract (involving 252 eyes) and type 2 diabetes mellitus received phacoemulsification cataract surgery in our hospital between January, 2017 and June, 2019. The patients were divided into group A (150 cases) and group B (102 cases) for cataract phacoemulsification with cumulative energy composite parameters of 8 and 10, respectively, and 90 nondiabetic patients received cataract phacoemulsification with a cumulative energy composite parameters of 10 served as the control. The macular thickness, best corrected visual acuity, visual acuity, and postoperative leakage in the 3 groups were evaluated at 1 week, 1 month, and 3 months after the surgery. RESULTS: The visual acuity was significantly improved after phacoemulsification better in all the 3 groups. At 3 months after the surgery, the proportions of patients with visual acuity ratio < 0.1 or >1.0, macular thickness, best corrected visual acuity and permeability differed significantly between groups A and B (P < 0.05), but not between group A and the control group (P > 0.05). At 1 month and 3 months after the surgery, the proportion of patients with visual acuity ratio < 0.1 was significantly lower and the rate of visual acuity ratio >1.0 was higher in group A than in group B. At 1 month after the operation, the total leakage rate in group A (31.1%) was higher than that in the control group (21.1%) but comparable with that in group B; at 3 months, the total leakage rates were significantly lower in group A than in group B (10.0% vs 32.4%, P < 0.05), and the leakage resulted mainly from local and diffuse permeation. CONCLUSIONS: Phacoemulsification can effectively improve the visual acuity of cataract patients especially in non-diabetic patients. A lower cumulative energy composite parameter achieves better outcomes in type 2 diabetic patients with cataract. The macular thickness, local infiltration and diffuse leakage can be used as indicators for assessing visual recovery and stabilization after phacoemulsification.

Modulation of transcriptional corepressor activity of prospero-related homeobox protein (Prox1) by SUMO modification.

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1's corepressor activity.

Characterization of a novel isoform of murine interferon regulatory factor 3.

Interferon (IFN) regulatory factors (IRFs) are a family of transcription mediators involved in the regulation of type I IFN (IFN-alpha/beta) transcription. Among the nine already identified IRFs, IRF-3 is a constitutively and ubiquitously expressed and plays a critical role in the transcriptional activation of type I IFN and IFN-stimulated genes including IFN-alpha1, IFN-beta and RANTES. In the present study, we report the identification of a novel alternatively spliced transcript of murine Irf-3 gene (mIrf-3) designated mIRF-3a. mIRF-3a is ubiquitously present in mouse tissues along with mIRF-3 and could be translocated into the nucleus in uninfected L929 cells. EMSA and reporter assay demonstrated that mIRF-3a binds to ISRE sequences in murine Ifn-beta promoter and represses Ifn-beta promoter activation induced by Newcastle disease virus infection. These results suggest that mIRF-3a may act as a modulator of mIRF-3 functions in vivo.

Evidence for inter- and intra-clade recombinations in rabies virus.

Homologous recombination is considered rare in negative-strand RNA viruses and has not been reported for rabies virus. In this study, full-length genomes of 44 rabies virus strains were analyzed for potential homologous recombination events. Phylogenetic analysis classified these strains into three clades. By applying six different recombination detection methods, one inter-clade and one intra-clade potential recombination events were identified with high confidence values. Software-predicted recombination break points of the two events were all located within the polymerase gene. This report presents the first evidence suggesting the possibility of homologous recombination in rabies virus, which could provide valuable insights for understanding the diversity and evolution of rabies virus as well as other negative-strand RNA viruses.

Identification of natural recombination in duck hepatitis B virus.

Due to its high similarity to human hepatitis B virus (HBV), duck HBV (DHBV) is often used as an important model for HBV research. While inter-genotypic recombination of HBV is common, it has not been reported with DHBV. In this study, 32 non-redundant DHBV complete genomes were analyzed using phylogenetic methods and classified into two clusters, corresponding to the 'Chinese' and 'Western country' branches previously reported based on geographical distribution. One 'Chinese' branch strain was isolated in Australia and three 'Western country' branch strains were isolated in China, suggesting cross-geographical distribution of both branches. Recombination analyses of the 32 DHBV genomes identified two possible inter-genotypic recombination events with high confidence value. These recombination events occurred between the lineages represented, respectively, by the Chinese isolate GD3 (AY536371, 'Chinese' branch) and the American isolate DHBV16 (K01834, 'Western country' branch), giving rise to two Chinese recombinant isolates CH4 (EU429324) and CH6 (EU429326). The identification of inter-genotypic recombination among circulating DHBV isolates suggests the usefulness of DHBV as a model for studying the mechanism of HBV recombination.

Development of novel therapeutics for chronic hepatitis B.

Chronic infection of hepatitis B virus (HBV) presents one of the serious public health challenges worldwide. Current treatment of chronic hepatitis B (CHB) is limited, and is composed of interferon and nucleoside/nucleotide reverse transcriptase inhibitors (NRTI). Interferon is poorly tolerated and is only responsive in a small fraction of CHB patients and NRTIs often face the problem of emergence of drug resistance during long-term treatment. The current treatment of CHB can be improved in several ways including genotyping mutations associated with drug resistance before treatment to guide the choice of NRTIs and suitable combinations among NRTIs and interferon. It is important to continue research in the identification of novel therapeutic targets in the life cycle of HBV or in the host immune system to stimulate the development of new antiviral agents and immunotherapies. Several antiviral agents targeting HBV entry, cccDNA, capsid formation, viral morphogenesis and virion secretion, as well as two therapeutic vaccines are currently being evaluated in preclinical studies or in clinical trials to assess their anti-HBV efficacy.

Prospero-related homeobox protein (Prox1) inhibits hepatitis B virus replication through repressing multiple cis regulatory elements.

Hepatitis B virus (HBV) gene transcription is controlled by viral promoters and enhancers, the activities of which are regulated by a number of cellular factors as well as virally encoded proteins. Negative regulation of HBV cis-element activities by cellular factors has been reported less widely than their activation. In this study, we report that nuclear factor Prospero-related homeobox protein (Prox1) represses HBV antigen expression and genome replication in cultured hepatocytes. By using reporter-gene analysis, three of the four HBV promoters, namely the enhancer II/core promoter (ENII/Cp), preS1 promoter (Sp1) and enhancer I/X promoter, were identified as targets for Prox1-mediated repression. Mechanistic analysis then revealed that, for ENII/Cp, Prox1 serves as a corepressor of liver receptor homologue 1 (LRH-1) and downregulates LRH-1-mediated activation of ENII/Cp, whereas for Sp1, Prox1 partially represses Sp1 activity by interacting directly with hepatocyte nuclear factor 1. Identification of Prox1 as an HBV repressor will help in the understanding of detailed interactions between viral cis elements and host cellular factors and may also form the basis for new anti-HBV intervention therapeutics.

Identification and characterization of peptides that interact with hepatitis B virus via the putative receptor binding site.

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.