Interaction of HnRNP F with the guanine-rich segments in viral antigenomic RNA enhances porcine reproductive and respiratory syndrome virus-2 replication.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (HnRNP) F is a member of HnRNP family proteins that participate in splicing of cellular newly synthesized mRNAs by specifically recognizing tandem guanine-tracts (G-tracts) RNA sequences. Whether HnRNP F could recognize viral-derived tandem G-tracts and affect virus replication remain poorly defined. METHODS: The effect of HnRNP F on porcine reproductive and respiratory syndrome virus (PRRSV) propagation was evaluated by real-time PCR, western blotting, and plaque-forming unit assay. The association between HnRNP F and PRRSV guanine-rich segments (GRS) were analyzed by RNA pulldown and RNA immunoprecipitation. The expression pattern of HnRNP F was investigated by western blotting and nuclear and cytoplasmic fractionation. RESULTS: Knockdown of endogenous HnRNP F effectively blocks the synthesis of viral RNA and nucleocapsid (N) protein. Conversely, overexpression of porcine HnRNP F has the opposite effect. Moreover, RNA pulldown and RNA immunoprecipitation assays reveal that the qRMM1 and qRRM2 domains of HnRNP F recognize the GRS in PRRSV antigenomic RNA. Finally, HnRNP F is redistributed into the cytoplasm and forms a complex with guanine-quadruplex (G4) helicase DHX36 during PRRSV infection. CONCLUSIONS: These findings elucidate the potential functions of HnRNP F in regulating the proliferation of PRRSV and contribute to a better molecular understanding of host-PRRSV interactions.

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling.

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/ß response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

HnRNP K reduces viral gene expression by targeting cytosine-rich sequences in porcine reproductive and respiratory syndrome virus-2 genome to dampen the viral growth.

HnRNP K is a well-known member of HnRNP family proteins that has been implicated in the regulation of protein expression. Currently, the impact of HnRNP K on the reproduction cycle of a broad range of virus were reported, while the precise function for PRRSV was lacking. In this study, we determined that both PRRSV infection and ectopic expression of N protein induced an enrichment of HnRNP K in the cytoplasm. Using RNA pulldown and RNA immunoprecipitation, we described the interactions between the KH2 domain of HnRNP K and cytosine-rich sequences (CRS) in PRRSV genomic RNA corresponding to Nsp7α coding region. Meanwhile, overexpression of HnRNP K inhibited viral gene expression and PRRSV replication, while silencing of HnRNP K resulted in an increased in virus yield. Taken together, this study assists in the understanding of PRRSV-host interactions, and the development of vaccines based on viral genome engineering.

Zinc Laurate Protects against Intestinal Barrier Dysfunction and Inflammation Induced by ETEC in a Mice Model.

Enterotoxigenic Escherichia coli (ETEC) infection is one of the most common bacterial causes of diarrhea in children and young farm animals. Medium-chain fatty acids (MCFAs) have been widely used for their antibacterial and immune functions. However, there is limited information regarding the role of MCFAs chelated with Zn in diarrhea induced by ETEC infection. Here, zinc laurate (ZnLa) was used to evaluate its protective effect in a mice diarrhea model induced by ETEC. A total of 45 ICR-weaned female mice were randomly assigned to marginal zinc deficiency (dZn), dZn, and ETEC infection groups (dZn+ETEC); ETEC infection was co-treated with a low, middle, or high dose of ZnLa (ZnLa LOW+ETEC, ZnLa MID+ETEC, and ZnLa HIGH+ETEC), respectively, to explore the effect and its mechanism of ZnLa on diarrhea and intestinal health of mice challenged with ETEC. To further compare the antibacterial efficiency of ZnLa and ZnSO4 in mice with ETEC infection, a total of 36 ICR-weaned female mice were randomly divided into ZnLa, ZnLa+ETEC, ZnSO4, and ZnSO4 and ETEC infection groups (ZnSO4+ETEC); moreover, the growth curve of ETEC also compared ZnLa and ZnSO4 in vitro. Mice pretreated with ZnLa were effectively guarded against body weight losses and increases in diarrhea scores induced by ETEC. ZnLa pretreatment also prevented intestinal barrier damage and ion transport in mice challenged with ETEC, as evidenced by the fact that the intestinal villus height and the ratio of villus height and crypt depth, tight junction protein, and Na+ absorption were higher, whereas intestinal permeability and anion secretion were lower in mice pretreated with ZnLa. In addition, ZnLa conferred effective protection against ETEC-induced intestinal inflammatory responses, as the increases in protein and mRNAs of proinflammatory cytokines were prevented in serum and jejunum, which was likely associated with the TLR4/MYD88/NF-κB signaling pathway. The increase in ETEC shedding and virulence-related gene expression was prevented in mice with ZnLa pretreatment. Finally, the growth of ETEC and virulence-related gene expression were lower in the ZnLa group than in ZnSO4 with an equal concentration of zinc. These findings suggest that ZnLa is a promising prevention strategy to remedy ETEC infection.

TRIM26-mediated degradation of nucleocapsid protein limits porcine reproductive and respiratory syndrome virus-2 infection.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, has ranked among the most economically important veterinary infectious diseases globally. Recently, tripartite motif (TRIMs) family members have arisen as novel restriction factors in antiviral immunity. Noteworthy, TRIM26 was reported as a binding partner of IRF3, TBK1, TAB1, and NEMO, yet its role in virus infection remains controversial. Herein, we showed that TRIM26 bound N protein by the C-terminal PRY/SPRY domain. Moreover, ectopic expression of TRIM26 impaired PRRSV replication and induced degradation of N protein. The anti-PRRSV activity was independent of the nuclear localization signal (NLS). Instead, deletion of the RING domain, or the PRY/SPRY portion, abrogated the antiviral function. Finally, siRNA depletion of TRIM26 resulted in enhanced production of viral RNA and virus yield in porcine alveolar macrophages (PAMs) after PRRSV infection. Overexpression of an RNAi-resistant TRIM26 rescue-plasmid led to the acquisition of PRRSV restriction in TRIM26-knockdown cells. Together, these data add TRIM26 as a potential target for drug design against PRRSV.

Effects of glycyrrhizin on the growth cycle and ATPase activity of PRRSV-2-infected MARC-145 cells.

Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and is devastating the swine industry. MARC-145 cells, an African green monkey kidney cell line, are sensitive to PRRSV-2, and are often used for in vitro studies on PRRSV-2. Preliminary research has shown that glycyrrhizin, an important active component extracted from traditional Chinese medicinal licorice, significantly inhibits the proliferation of PRRSV-2 in MARC-145 cells; however, the in-depth molecular mechanism remains unclear. By determining the cell growth cycle, this study found that PRRSV-2 infection first increased the content of G1-phase MARC-145 cells and then decreased the content of G1-phase cells. Moreover, glycyrrhizin affected the role of PRRSV-2 in regulating the cell cycle. Furthermore, PRRSV-2 had the highest proliferation titer in G0/G1-phase MARC-145 cells, and glycyrrhizin reduced the content of PRRSV-2 in synchronized MARC-145 cells. According to the results of ATPase detection, PRRSV-2 infection weakened the Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in MARC-145 cells, while glycyrrhizin significantly enhanced their activities in PRRSV-2-infected MARC-145 cells. The above results provide theoretical support toward clarifying the mechanism by which glycyrrhizin inhibits the proliferation of PRRSV-2 in MARC-145 cells. Moreover, these results offer references for the development and use of glycyrrhizin and the clinical treatment of PRRSV-2 infection.

pH and charge reversal-driven nanoplatform for efficient delivery of therapeutics.

Nanomedicine which delivers therapeutics to tumours holds great potential for cancer treatment. However, endosomal trapping and uncontrollable release usually limit the efficiency of nanomedicine. Herein, a smart mesoporous silica based nanoplatform was constructed, in which mesoporous silica nanoparticles (MSNs) serve as the core, capped with pH-induced charge-reversal polymer -PAH-cit- and cationic polyelectrolyte polyethyleneimine (PEI). The oppositely charged polymer can not only act as a gatekeeper for controlled release, but also mediated efficient endosomal escape of the therapeutics. Under the acidic endosomal environment, the hydrolysis of acid-cleavable bonds in PAH-Cit would trigger the charge reversal and endosomal escape of the nanoplatform for efficient drug release. Furthermore, the prepared nanoplatform demonstrated a higher tumor cell proliferation inhibition rate than free theruputics in vitro assays and significantly inhibited tumour growth in the 4T1 tumour model in mice. Therefore, our strategy offers a simple and general nanoplatform to delivery therapeutics to tumours with efficient endosomal escape and controlled release.

A chromosome-scale genome assembly of Antheraea pernyi (Saturniidae, Lepidoptera).

Antheraea pernyi is a semi-domesticated lepidopteran insect species valuable to the silk industry, human health, and ecological tourism. Owing to its economic influence and developmental properties, it serves as an ideal model for investigating divergence of the Bombycoidea super family. However, studies on the karyotype evolution and functional genomics of A. pernyi are limited by scarce genomic resource. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first high-quality A. pernyi genome from a single male individual. The genome is 720.67 Mb long with 49 chromosomes and a 13.77-Mb scaffold N50. Approximately 441.75 Mb, accounting for 60.74% of the genome, was identified as repeats. The genome comprises 21,431 protein-coding genes, 85.22% of which were functionally annotated. Comparative genomics analysis suggested that A. pernyi diverged from its common ancestor with A. yamamai ~30.3 million years ago, and that chromosome fission contributed to the increased chromosome number. The genome assembled in this work will not only facilitate future research on A. pernyi and related species but also help to progress comparative genomics analyses in Lepidoptera.

TRIM59 inhibits porcine reproductive and respiratory syndrome virus (PRRSV)-2 replication in vitro.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV), has ranked among the major economically significant pathogen in the global swine industry. The PRRSV nonstructural protein (nsp)11 possesses nidovirus endoribonuclease (NendoU) activity, which is important for virus replication and suppression of the host innate immunity system. Recent proteomic study found that TRIM59 (tripartite motif-containing 59) interacted with the nsp11, albeit the exact role it plays in PRRSV infection remains enigmatic. Herein, we first confirmed the interaction between nsp11 and TRIM59 in co-transfected HEK293T cells and PRRSV-infected pulmonary alveolar macrophages (PAMs). The interacting domains between TRIM59 and nsp11 were further determined to be the N-terminal RING domain in TRIM59 and the C-terminal NendoU domain in nsp11, respectively. Moreover, we reported that overexpression of TRIM59 inhibited PRRSV infection in Marc-145 cells. Conversely, small interfering RNA (siRNA) depletion of TRIM59 resulted in enhanced production of PRRSV in PAMs. Together, these data add TRIM59 as a crucial antiviral component against PRRSV and provide new insights for development of new compounds to reduce PRRSV infection.

Suppression of porcine reproductive and respiratory syndrome virus proliferation by glycyrrhizin.

Glycyrrhizin is a natural component extracted from the roots of Glycyrrhiza glabra. In this study, we investigated the antiviral activity of glycyrrhizin against porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Our results showed that treatment with glycyrrhizin significantly reduced PRRSV proliferation and PRRSV-encoded protein expression in a dose-dependent manner. Mechanistically, glycyrrhizin mainly inhibits the penetration stage, and has little effect on the steps of adsorption or release of PRRSV in its life cycle. Furthermore, we were able to exclude a direct inhibitory action of glycyrrhizin on PRRSV particles. Given these results, glycyrrhizin may be a candidate component for a novel porcine reproductive and respiratory syndrome (PRRS) control strategy.

Porcine reproductive and respiratory syndrome virus infection triggers HMGB1 release to promote inflammatory cytokine production.

The high mobility group box 1 (HMGB1) protein is an endogenous damage-associated molecular pattern (DAMP) molecule involved in the pathogenesis of various infectious agents. Based on meta-analysis of all publicly available microarray datasets, HMGB1 has recently been proposed as the most significant immune modulator during the porcine response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, the function of HMGB1 in PRRSV pathogenesis is unclear. In this study, we found that PRRSV infection triggers the translocation of HMGB1 from the nucleus to the extracellular milieu in MARC-145 cells and porcine alveolar macrophages. Although HMGB1 has no effect on PRRSV replication, HMGB1 promotes PRRSV-induced NF-κB activation and subsequent expression of inflammatory cytokines through receptors RAGE, TLR2 and TLR4. Our findings show that HMGB1 release, triggered by PRRSV infection, enhances the efficiency of virus-induced inflammatory responses, thereby providing new insights into the pathogenesis of PRRSV infection.

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China.

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003-2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003-2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.

Ligation of Fc gamma receptor IIB enhances levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto its host cells, such as monocytes and macrophages, through Fc receptor-mediated endocytosis. FcγRIIB is the only inhibitory receptor with a tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, where counters the "ITAM triggered" activation signals and down-regulates phagocytosis. However, porcine FcγRIIB's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRIIB in PAM cells up-regulated significantly mRNA levels of IFN-α and TNF-α at any time point post-pretreatment, suggesting that porcine FcγRIIB signal can enhance the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRIIB indicated that selective activation of porcine FcγRIIB in PAM cells enhanced mRNA levels of antiviral cytokine (IFN-α and TNF-α) and repressed mRNA levels of IL-10 in response to PRRSV infection, suggesting that FcγRIIB ligation can enhance the antiviral immune response to PRRSV infection. In addition, FcγRIIB ligation to infection indicated that PRRSV replication in PAM was not positive correlation with increasing of IFN-α mRNA levels and decreasing of IL-10 mRNA levels, suggesting that there is complex viral replication mechanism in immune cells such as PAM for PRRSV.

Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing.

Receptors for the Fc portion of IgG (FcγRs) are expressed on various leukocytes and they modulate both humoral and cell-mediated immune responses with different capacities for IgG binding and phagocytosis. Four different types of FcγRs, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibitory FcγRIIb) in humans, one isoform (inhibitory FcγRIIb) in mice, and two isoforms (inhibitory FcγRIIb and activating FcγRIIc) in cattle. Two alternativly spliced isoforms of FcγRIIb, b1 and b2, have been identified in humans, mice and cattle, however, only two porcine FcγRIIb transcripts have been reported. In this study, we report the identification of three new porcine FcγRIIb transcript and analyze the sequences of five porcine FcγRIIb transcript generated by alternative splicing. The porcine transcript 1 and porcine transcript 2 have a high homology and structural similarity with human b1 and b2, respectively, while there is only one alanine residue difference at the signal peptide region between porcine transcript 1 and transcript 4, as well as porcine transcript 2 and transcript 3. This is the first time that an alternativly spliced isoform of porcine transcript 5 is described in pigs rather than humans or other animals. All the five transcripts have the consensus sequence of an ITIM (ITYSLL) in their cytoplasmic tails. Analysis results indicate that the five transcripts serve as inhibitory receptors and are these sub-isoforms or alternativly spliced isoforms. Immunoglobulin-binding assays show that transcript 1, transcript 2, transcript 3 and transcript 4 have binding activity for IgG immune complexes, whereas transcripts 5 without domain 2 can not bind IgG-complexes. It is now clear that porcine FcγRIIb exists as five sub-isoforms at least. These sub-isoforms may individually modulate FcγRIIb-mediated immune responses in the porcine immune system.

Molecular cloning and characterization of a porcine Fc gamma RIIb sub-isoform(FcγRIIb1).

Receptors for the Fc fragment of IgG (FcγRs) constitute one of the main effector mechanisms through which IgG immune complexes exert their action. Four FcγRs, FcγRI (CD64) with high affinity, FcγRI with intermediate affinity, FcγRII (CD32) and FcγRIII (CD16) with low affinity, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibiting FcγRIIb) existing in humans, one isoform in mice (inhibiting FcγRIIb), and two isoforms in cattle (inhibiting FcγRIIb, activating FcγRIIc). Two splice sub-isoforms of FcγRIIb, FcγRIIb1(b1) and FcγRIIb2(b2), have been identified in humans, mice and cattle, however, few of FcγRIIb sub-isoforms have been investigated in pig. In this study, we describe the molecular cloning, sequencing and characterization of a porcine FcγRIIb sub-isoform, FcγRIIb1. The cDNA encoding porcine FcγRIIb1 was isolated from peripheral blood leucocytes RNA with RT-PCR. The porcine FcγRIIb1 cDNA contains a 951bp open-reading frame, encoding a 316 amino acid transmembrane glycoprotein composed of two immunoglobulin (Ig)-like extracellular domains, a transmembrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibiting motif (ITIM). The porcine FcγRIIb1 shares 98.3% homology and has a 19 amino acid in-frame insertion in cytoplasmic tail when compared with amino acid sequence of DQ026064. Immunofluorescence analysis showed that the glycoprotein encoded by the porcine FcγRIIb1 cDNA was expressed in the stable transfected COS-7 cells, and an immunoglobulin-binding assay showed that it had binding activity for IgG immune complexes. Identification of the porcine FcγRIIb1 will help our understanding of the molecular basis of IgG-FcγR interaction in the porcine immune response.