Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon gene and characterisation of its expression in response to nodavirus infection

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6–7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.     

Characterization of virus/double-stranded RNA-dependent induction of antimicrobial peptide hepcidin in trout macrophages

Hepcidin is an antimicrobial peptide responsive to bacterial infection. We report the characterization of a virus/double-stranded RNA (dsRNA) induction of hepcidin in rainbow trout (Oncorhynchus mykiss). Increased level of hepcidin mRNA was observed in trout macrophage RTS11 cells treated with polyinosinic–polycytidylic acid (poly I:C), a mimic of viral dsRNA. The induction was also observed in poly I:C-injected trout, demonstrating that it is a bona fide biological response. The induction was not observed in livers or hepatic RTH1B2C cells despite the presence of IFN response. The induction required de novo protein synthesis. Studies on the kinetic relationship among the poly I:C-regulated hepcidin induction and IFN response indicated that the two responses were uncoupled. Interestingly, in RTS11 infected with infectious hematopoietic necrosis virus, the level of hepcidin was increased as expected but subsequently reduced to below baseline as the infection progressed, whereas IFNs, Mx1 and TLR3 were still increasing.     

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.     

Cellular localization and function of the antiviral protein, ovine Mx1 (oMx1): I. Ovine Mx1 is secreted by endometrial epithelial cells via an 'unconventional' secretory pathway

PROBLEM: Embryonic loss is a major contributor to infertility. Understanding factors contributing to embryonic loss will aid in development of technologies to improve/regulate fertility in animals and humans. METHOD OF STUDY: We tested the hypothesis that the antiviral protein, ovine Mx1 (oMx1), is secreted by uterine epithelial cells. Uterine flushes were obtained from cyclic and early pregnant ewes and examined for levels of oMx1 protein. The pathway for ovine Mx1 secretion in ovine glandular epithelial (oGE) cells was determined using brefeldin A (BFA), an inhibitor of the conventional secretory pathway. Effects of BFA were determined using beta2-microglobulin (beta2MG) as a marker for the conventional secretory pathway, and interferon stimulated gene 15 (ISG15) and Galectin-1 (Gal-1) as markers for the unconventional secretory pathways. RESULTS: Ovine Mx1 protein levels were low in uterine flushes from cyclic ewes and levels increased in pregnant ewes after D 15. Ovine GE cells secreted oMx1 in response to interferon and secretion was not reduced by BFA, suggesting oMx1 was secreted via an unconventional secretory pathway. beta2MG secretion was reduced by BFA, whereas ISG15 and Gal-1 were not. CONCLUSION: This is the first report that the antiviral protein, oMx1, is secreted and provides evidence that secretion occurs via unconventional secretory pathway(s).     

Effects of polyinosinic-polycytidylic acid (Poly I:C) on naloxone-precipitated withdrawal in morphine-dependent mice

Viral infections are frequently found in opioid addicts, subjecting them to immune challenge. However, the effects of immune challenge on opioid withdrawal are not fully understood. In the present study, mice were intraperitoneally injected with 2mg/kg polyinosinic-polycytidylic acid (Poly I:C, a viral mimetic) for 3 days to induce an immune challenge, followed by subcutaneous injection of morphine 3 times per day for 3 days to induce morphine dependence. Withdrawal was induced by an intraperitoneal injection of 5mg/kg naloxone, an opioid receptor antagonist. The results showed that Poly I:C pretreatment did not alter body weight loss, jumping behavior, or locomotion during naloxone-precipitated withdrawal. In contrast, Poly I:C pretreatment significantly increased immobility time in the tail suspension test. Our findings suggest that Poly I:C-induced immune challenge has no effects on acute physical opioid withdrawal symptoms but facilitates depression-like behavior.     

Characterization of a membrane protein (VP001L) from infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of megalocytivirus, Iridoviridae. A novel membrane protein corresponding to the first open reading frame (ORF001L) of ISKNV genome was identified. This 378-residue protein, termed the VP001L protein, has a high content of hydrophobic sequences and contains 10–11 putative transmembrane domains, indicating it may be a membrane protein. The VP001L mRNA start site was extended 433 bp upstream of the start codon and the temporal analysis showed that the VP001L gene was first transcribed at 8 h post-infection (h.p.i.). VP001L protein was detected on the plasma membrane of ISKNV infected cells by immunofluresence. In order to further investigate different transmembrane domains’ influence on subcellular localization of VP001L, series of truncated or deleted mutants were constructed with GFP at the C terminus. The transfection results indicated that the second putative transmembrane domain played a determinative role in VP001L’s membrane localization and the translocation of the first and third transmembrane domains depended on their interactions with the second one. Therefore, this novel VP001L protein is considered to serve as a model for analyzing the topology and roles of different hydrophobic regions in multi-transmembrane proteins.     

Identification of a nonconventional motif necessary for the nuclear import of the human parvovirus B19 major capsid protein (VP2)

Human parvovirus B19 replicates and encapsidates its genome in the nucleus of erythroid progenitors in vivo and in vitro. We wanted to understand the determinants necessary for the nuclear transport of the major coat protein, VP2, which makes up about 96% of the viral capsid proteins. A nonconsensus basic motif, KLGPRKATGRW, necessary for the nuclear localization of VP2 was identified and shown to be able to import reporter proteins into the nucleus. The sequence is conserved among the VP2 C-terminal region of erythroviruses. This newly identified sequence will facilitate the understanding of the replication of these viruses.     

Cellular localization and function of the antiviral protein, ovine Mx1 (oMx1): II. The oMx1 protein is a regulator of secretion in an ovine glandular epithelial cell line

PROBLEM: Embryonic loss is a major contributor to infertility. Understanding factors affecting embryonic loss will help increase fertility. METHOD OF STUDY: We investigated if ovine Mx1 (oMx1) mediated secretion by ovine glandular epithelial (oGE) cells using small interfering RNA (siRNA). Effects on secretion were examined through the conventional endoplasmic reticulum-Golgi pathway using beta2- microglobulin (beta2MG) as a marker, and interferon-stimulated gene 15 (ISG15) as a marker for unconventional secretion. RESULTS: Mx1 siRNA reduced oMx1 mRNA levels at 12 and 24 hr after IFN-tau treatment (P < 0.05), without affecting levels of oMx2, ISG15, 2',5'-oligoadenylate synthetas or beta2MG. Mx1 siRNA reduced Mx1 protein levels at 48 and 120 hr after treatment (P < 0.05) and protein levels remained low at 120 hr. Transient oMx1 knock-down reduced secretion of oMx1 (P < 0.01). ISG15 protein in secretions was reduced without affecting intracellular levels (P < 0.05). Levels of beta2MG in secretions were not affected by Mx1 siRNA. CONCLUSION: We showed that oMx1 protein is secreted by oGE cells and that reduction in oMx1 protein levels by siRNA reduced secretion of ISG15, but not beta2MG. Results support the hypothesis that oMx1 is a regulator of secretion through unconventional secretory pathway(s).     

Formulations combining CpG containing oliogonucleotides and poly I:C enhance the magnitude of immune responses and protection against pancreas disease in Atlantic salmon

Both CpG oligodeoxynucleotides and double-stranded RNA (poly I:C) have documented effects as treatments against several viral diseases in fish. However, as stand-alone treatments their effects have been modest. We have tested here whether CpG and poly I:C, alone or in combination induce protection against Salmonid Alphavirus (SAV), the causative agent of pancreas disease in Atlantic salmon. Our results revealed a significant reduction of viraemia 2 weeks after ip injection of the combined treatment and 1 week after challenge with SAV subtype 3, followed by reduced SAV induced heart pathology 3 weeks later. The SAV titers in blood samples from the combination group were lower as compared to single treatments with either CpG or poly I:C. Surprisingly, reduced SAV levels could also be found in fish as long as 7 weeks after receiving the combination treatment. The expression of IFNγ and to a lesser extent IFNa and Mx was up-regulated in head kidney and spleen 5 days after the fish had been treated with CpG and poly I:C. Furthermore, the complement factor C4 was depleted in serum 8 weeks post treatment, suggesting complement activation leading to C4 consumption. We hypothesize that the CpG/poly I:C-induced protection against SAV3 is mediated by mechanisms involving type I and type II IFN induced antiviral activity and complement mediated protective responses.     

Poly I:C primes the suppressive function of human palatine tonsil-derived MSCs against Th17 differentiation by increasing PD-L1 expression

It has been established that mesenchymal stem cells (MSCs) can have a suppressive effect on T cells, yet much remains unknown about the underlying mechanisms that support this effect. The T cell co-stimulatory pathway involving the programmed death-1 (PD-1) receptor and its ligand PD-L1 regulates T cell activation, tolerance, and subsequent immune-mediated tissue damage. In this study, human palatine tonsil-derived MSCs (T-MSCs) constitutively expressed PD-L1 and exhibited a suppressive activity that specifically targeted murine Th17 differentiation. Additionally, polyinosinic–polycytidylic acid (poly I:C), a Toll-like receptor 3 (TLR3) ligand, increased PD-L1 expression on T-MSCs. The elevated PD-L1 levels enhanced the suppressive functions of T-MSCs on Th17 differentiation. Therefore, pre-stimulation of T-MSCs with poly I:C may serve as an effective therapeutic priming step for modulating Th17-dominant immune responses.     

Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNAβ satellite

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of geminiviruses. Capsid proteins (CPs) of geminiviruses are multifunctional proteins thought to be involved in this process. The CP of monopartite geminiviruses is absolutely essential for virus movement. To more precisely examine the role of CP, we have constructed a series of single and double deletions into the coding sequence of Tomato leaf curl Java virus (ToLCJAV) CP and examined sub-cellular localization using transient expression of GFP fusion proteins. In this report, the domains of the CP encoded by ToLCJAV localized in the nucleus/nucleolus and cytoplasm in transfected cells were mapped. Deletion analysis revealed that the Arg-rich cluster from amino acids (aa) ¹⁶KVRRR²⁰ in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). The region from aa ⁵²RKPR⁵⁵ contained basic amino acid cluster was capable to redirect the CP to the nucleus. Further, both transient expression and yeast hybrid assays demonstrated that CP was capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa (²⁴⁵LKIRIY²⁵⁰) reside at C-terminal part of CP. This hydrophobic region caused transport of GFP to the cytoplasm. However, ToLCJAV CP NLSs and NES show peculiarities in the number and position of basic residues. Taken together, these results demonstrated that ToLCJAV CP shuttles between the nucleus and cytoplasm, such an activity homolog to bipartite geminivirus BV1 ORF.     

Characterization of the role of TCRγδ in NK cell accumulation during viral liver inflammation

Polyinosinic-polyctidylic acid (Poly I:C) is a viral RNA mimic that can induce immune responses similar to that seen during viral infection. Although poly I:C administration into mice is associated increased NK cell infiltrates in the liver, the mechanisms underlying increased hepatic NK cell accumulation in response to poly I:C administration are incompletely defined. In the current study, we have identified a novel and important role for γδT cells in driving the accumulation and activation of NK cells in the liver during poly I:C-mediated viral liver infection. Specifically, NK cell accumulation but not activation in γδT cell deficient mice following poly I:C administration was significantly attenuated in comparison to that seen in poly I:C-treated wildtype mice. The ability of γδT cells to promote NK cell accumulation and activation in the liver may be virus-specific since NK cell accumulation in the liver was not altered by TCRγδ deficiency following adenovirus administration.