类泛素修饰蛋白bISG15抗血清在体外修饰系统中的应用

ISG15由干扰素刺激基因15编码,是最早被发现的类泛素修饰分子.病毒感染以及干扰素刺激可以强烈诱导其表达.与泛素类似,ISG15可以共价连接到其他蛋白分子上进行修饰,但ISG15及其连接修饰的功能作用还有很多尚未知.最近的研究表明,ISG15及其修饰作用在先天免疫中起着重要的作用.将牛类ISG15基因克隆进入pET28a(+)原核表达载体,并且表达了可溶的融合有His-tag标签的bISG15融合蛋白.使用Ni-NTA葡聚糖进行纯化浓缩.纯化蛋白免疫Balb/c小鼠并获得抗血清.Western印迹实验显示,抗血清可以特异地识别在真核细胞中表达的bISG15.浓缩的bISG15以及制备的抗血清用于建立bISG15的体外修饰系统.实验证明,使用该系统bISG15可以连接到细胞蛋白上进行修饰.     

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.     

Alpha-interferon in Kikuchi’s disease

In Japan, histiocytic necrotizing lymphadenitis (Kikuchi’s disease) is a relatively common reactive lesion affecting lymph nodes, but the histogenesis and pathogenesis of the disease have not been clarified. Alpha-interferon has a role in the body’s defense against, viral infections. Using a polyclonal antibody to human alpha-interferon, we found numerous cells, mainly histiocytes, containing alpha-interferon in affected foci in the lymph nodes from 24 patients with Kikuchi’s disease. Tubuloreticular structures, thought by some authors to be associated with the production of interferon, were detected by electron microscopy in histiocytes, activated lymphocytes and vascular endothelial cells in the affected foci. These results suggested that the formation of tubuloreticular structures is a secondary phenomenon following stimulation by alpha-interferon. Further, the activity of 2′–5′ oligoadenylate synthetase, which is induced by alpha-interferon and enhanced during the early or active stage of viral infection, showed increased levels of activity in the active stage of Kikuchi’s disease and decreased to normal levels in the convalescent stage 2 weeks later. These results suggested the possibility of a viral etiology for Kikuchi’s disease.     

A neutrophil-derived antiviral protein: induction requirements and biological properties

Polymorphonuclear neutrophilic granulocytes (PMN) have been implicated as playing a role in antiviral defense. In addition to having phagocytic and cytotoxic activities, PMN may produce an antiviral substance with interferon (IFN)-like activity. The product, for which the name polyferon (PF) has been coined, is produced upon direct encounter of PMN with bovine herpesvirus 1 (BHV-1)-infected bovine cells or membranes thereof. Exposure to purified virus only does not induce PF. The intimate interaction between PMN and the membranes was also revealed by electron microscopy studies. Bovine cells infected with herpes simplex virus type 1 could also induce PF production by bovine PMN, whereas cells infected with BHV-2, herpes simplex virus type 2, equine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus, or parainfluenza virus 3 were unable to do so. Results obtained in experiments using transfected cells expressing BHV-1 glycoproteins as well as blocking experiments using BHV-1 glycoprotein-monospecific antibodies suggested that a combination of both viral product(s) and host cell factor(s) unique to bovine cells is required for induction of PF production by PMN. PF, which appeared in detectable amounts 12 to 18 h after exposure of PMN to the appropriate inducer, could not be neutralized by antibodies to bovine IFN-alpha, -beta, and -gamma. PF may nevertheless belong to the IFN family of proteins, as indicated by its ability to induce 2',5'-oligoadenyl synthetase in various cell types that are responsive to bovine IFNs and by its antiviral spectrum. It does, however, differ from the other cytokines in most immunological characteristics tested so far, including major histocompatibility complex class II antigen induction, cell migration, and cytotoxicity.     

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.     

Glycoprotein IV of bovine herpesvirus 1-expressing cell line complements and rescues a conditionally lethal viral mutant.

Glycoprotein IV (gIV) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus glycoprotein D, represents a major component of the viral envelope and a dominant immunogen. To analyze the functional role of gIV during BHV-1 replication, cell line BUIV3-7, which constitutively expresses gIV, was constructed and used for the isolation of gIV- BHV-1 mutant 80-221, in which the gIV gene was replaced by a lacZ expression cassette. On complementing gIV-expressing cells, the gIV- BHV-1 replicated normally but was unable to form plaques and infectious progeny on noncomplementing cells. Further analysis showed that gIV is essential for BHV-1 entry into target cells, whereas viral gene expression, DNA replication, and envelopment appear unchanged in both noncomplementing and complementing cells infected with phenotypically complemented gIV- BHV-1. The block in entry could be overcome by polyethylene glycol-induced membrane fusion. After passaging of gIV- BHV-1 on complementing cells, a rescued variant, BHV-1res, was isolated and shown to underexpress gIV in comparison with its wild-type parent. Comparison of the penetration kinetics of BHV-1 wild type, phenotypically complemented gIV- BHV-1, and BHV-1res indicated that penetration efficiency correlated with the amount of gIV present in virus particles. In conclusion, we show that gIV of BHV-1 is an essential component of the virion involved in virus entry and that the amount of gIV in the viral envelope modulates the penetration efficiency of the virus.     

Role of interferon-gamma in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells

In the present study, the possible role of interferon (IFN)-gamma on the induction of cytotoxic activity of peripheral blood mononuclear leukocytes (PBML) from BHV-1-immune cattle was investigated. Supernatants obtained from BHV-1-immune PBML, stimulated under conditions similar to those required to demonstrate cytotoxicity, contained an antiviral substance capable of inducing 2'-5'-oligoadenylate synthetase activity in MDBK cells and MHC class II antigen expression on epithelial cells. These supernatants also contained IFN-alpha, but were devoid of tumor necrosis factor and interleukin-2 biological activities. Further studies during primary infection and hyperimmunization with BHV-1 showed that IFN-gamma production and non-MHC-restricted cytotoxicity against BHV-1-infected targets always occurred concomitantly, suggesting that they represent an important part of the detectable CMI responses mounted against this virus. Furthermore, it was also demonstrated that cytotoxicity of PBML against BHV-1-infected cells was reduced with the addition of antibodies to bovine IFN-gamma to the cytotoxic assay. Bovine recombinant IFN-gamma was able to enhance the in vitro cytotoxic activity of PBML from immune cattle, but not from their nonimmune counterparts. This suggests that other factors, in addition to IFN-gamma, may be essential in the development of non-MHC-restricted cytotoxic responses during BHV-1 infection.     

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.     

APOBEC3A is a specific inhibitor of the early phases of HIV-1 infection in myeloid cells

Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for viral spread and as a viral reservoir. Yet, cells of this lineage generally resist HIV-1 infection when compared to cells of other lineages, a phenomenon particularly acute during the early phases of infection. Here, we explore the role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that is highly expressed in myeloid cells, but so far lacks a known antiviral effect against retroviruses. Using ectopic expression of APOBEC3A in established cell lines and specific silencing in primary macrophages and dendritic cells, we demonstrate that the pool of APOBEC3A in target cells inhibits the early phases of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1, specifically in cells of myeloid origins. In these cells, APOBEC3A affects the amount of vDNA synthesized over the course of infection. The susceptibility to the antiviral effect of APOBEC3A is conserved among primate lentiviruses, although the viral protein Vpx coded by members of the SIV(SM)/HIV-2 lineage provides partial protection from APOBEC3A during infection. Our results indicate that APOBEC3A is a previously unrecognized antiviral factor that targets primate lentiviruses specifically in myeloid cells and that acts during the early phases of infection directly in target cells. The findings presented here open up new venues on the role of APOBEC3A during HIV infection and pathogenesis, on the role of the cellular context in the regulation of the antiviral activities of members of the APOBEC3 family and more generally on the natural functions of APOBEC3A.     

CCAAT/enhancer binding protein-beta negatively regulates the expression of glycerol-3-phosphate dehydrogenase 1 in pig PK-15 cells

Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5’- TKNNGCAAK -3’). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5’-TKNNGCAAK -3’). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells.     

Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.     

Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 μM for DENV-2 and 1.3 μM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.