Inhibition of Polyinosinic-Polycytidylic Acid-Induced Acute Pulmonary Inflammation and NF-κB Activation in Mice by a Banana Plant Extract.

NF-κB activation is pivotal for the excess inflammation causing the critical condition and mortality of respiratory viral infection patients. This study was aimed to evaluate the effect of a banana plant extract (BPE) on suppressing NF-κB activity and acute lung inflammatory responses in mice induced by a synthetic double-stranded RNA viral mimetic, polyinosinic-polycytidylic acid (poly (I:C)). The inflammatory responses were analyzed by immunohistochemistry and HE stains and ELISA. The NF-κB activities were detected by immunohistochemistry in vivo and immunofluorescence and Western blot in vitro. Results showed that BPE significantly decreased influx of immune cells (neutrophils, lymphocytes, and total WBC), markedly suppressed the elevation of pro-inflammatory cytokines and chemokines (IL-6, RANTES, IFN-γ, MCP-1, keratinocyte-derived chemokine, and IL-17), and restored the diminished anti-inflammatory IL-10 in the bronchoalveolar lavage fluid (BALF) of poly (I:C)-stimulated mice. Accordingly, HE staining revealed that BPE treatment alleviated poly (I:C)-induced inflammatory cell infiltration and histopathologic changes in mice lungs. Moreover, immunohistochemical analysis showed that BPE reduced the pulmonary IL-6, CD11b (macrophage marker), and nuclear NF-κB p65 staining intensities, whilst restored that of IL-10 in poly (I:C)-stimulated mice. In vitro, BPE antagonized poly(I:C)-induced elevation of IL-6, nitric oxide, reactive oxygen species, NF-κB p65 signaling, and transient activation of p38 MAPK in human lung epithelial-like A549 cells. Taken together, BPE ameliorated viral mimic poly(I:C)-induced acute pulmonary inflammation in mice, evidenced by reduced inflammatory cell infiltration and regulation of both pro- and anti-inflammatory cytokines. The mechanism of action might closely associate with NF-κB signaling inhibition.

Virion Secretion of Hepatitis B Virus Naturally Occurring Core Antigen Variants.

In natural infection, hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations. The most frequent HBc variant in chronic hepatitis B patients is mutant 97L, changing from an isoleucine or phenylalanine to a leucine (L) at HBc amino acid 97. One dogma in the HBV research field is that wild type HBV secretes predominantly virions containing mature double-stranded DNA genomes. Immature genomes, containing single-stranded RNA or DNA, do not get efficiently secreted until reaching genome maturity. Interestingly, HBc variant 97L does not follow this dogma in virion secretion. Instead, it exhibits an immature secretion phenotype, which preferentially secretes virions containing immature genomes. Other aberrant behaviors in virion secretion were also observed in different naturally occurring HBc variants. A hydrophobic pocket around amino acid 97 was identified by bioinformatics, genetic analysis, and cryo-EM. We postulated that this hydrophobic pocket could mediate the transduction of the genome maturation signal for envelopment from the capsid interior to its surface. Virion morphogenesis must involve interactions between HBc, envelope proteins (HBsAg) and host factors, such as components of ESCRT (endosomal sorting complex required for transport). Immature secretion can be offset by compensatory mutations, occurring at other positions in HBc or HBsAg. Recently, we demonstrated in mice that the persistence of intrahepatic HBV DNA is related to virion secretion regulated by HBV genome maturity. HBV virion secretion could be an antiviral drug target.

Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model.

Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild-type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant, I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants, I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: (i) excessive numbers of serum HBV virions containing immature genomes, (ii) significantly reduced numbers of intracellular relaxed-circle and single-stranded DNAs, and (iii) less persistent intrahepatic and secreted HBV DNAs than wild-type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model.IMPORTANCE Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.

Immunocompetent and Immunodeficient Mouse Models for Enterovirus 71 Pathogenesis and Therapy.

Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death. At present, no effective treatment for EV71 is available. We reviewed here various mouse models for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains. To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71 clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate viral entry in cell culture. However, in vivo infection with human SCARB2 receptor transgenic mice appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors, immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed in models deficient in various arms of innate or humoral immunity. These models are being actively used for antiviral research.

A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71.

Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis.

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

UNLABELLED: Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE: In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.

Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD).

The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176) and ARD I-III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I-IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.

Nuclear export and import of human hepatitis B virus capsid protein and particles.

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1(Delta cub/Delta cub)) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7-9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1(Delta cub/Delta cub) embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.