Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.     

Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2

The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections.     

The structural proteins of Newcastle disease virus. I. Identification of a minor internal protein

The nature of the G-loop heterogeneity and of the split end heterogeneity in Mu phage DNA have been studied. The structures of the molecules seen in self-renatured Mu phage DNA samples show that there are two classes of native molecules in which the G sequence occurs either in a direct or in an inverted order. By studying the structure of a single strand of Mu DNA when spread under weakly denaturing conditions, it is shown that the G region is flanked by very short (50 nucleotides or less) inverted repeat sequence. The G loop inversion is attributed to a phage enzyme-directed reciprocal recombination between these inverted repeat sequences. A second slightly larger inverted repeat sequence occurs within the G loop, but this sequence is not active in reciprocal recombination. By studying suitable diheteroduplexes of F-prime factors containing an inverted Mu prophage, with a reference F factor and with Mu phage DNA, it is shown that the highly heterogeneous split ends of Mu phage are lost when Mu becomes a prophage.     

PRRSV灭活抗原免疫接种后抗主要结构蛋白抗体的产生规律研究

目的检测PRRSV灭活抗原免疫接种后抗主要结构蛋白抗体的产生规律。方法用差速离心法纯化PRRSV Hn-3/06株,0.1%甲醛灭活PRRSV,加入弗氏佐剂乳化制备成PRRSV灭活抗原。20只昆明系小鼠(7～8周龄)随机分为2组,每组10只,每次每只110μg蛋白剂量,第1组免疫1次,第2组免疫2次,每次间隔7 d。每隔7 d采血1次,间接ELISA方法检测血清中抗PRRSV及GP5、M和N蛋白的抗体效价。用Marc-145细胞进行细胞中和试验,间接免疫荧光灶抑制方法检测血清中和抗体效价。结果 2组小鼠均产生针对PRRSV及各结构蛋白的抗体,抗PRRSV的抗体和抗N蛋白的抗体一免后14 d出现,21 d后抗体效价达到1∶1 280,35 d抗体水平达到最高;抗M蛋白的抗体一免后42 d出现,49 d抗体水平达到最高,抗体效价达到1:160;抗GP5蛋白的抗体一免后49 d出现,至56 d抗体水平呈上升趋势,抗体效价达到1∶80。免疫2次的小鼠各项ELISA抗体效价总体略微偏高。细胞中和试验结果表明第1组免疫小鼠免疫后42 d左右产生微弱的中和抗体;第2组免疫小鼠一免后35～49 d产生可检测到的中和抗体,42 d时中和抗体水平达到最高,抗体效价达到1∶2。结论纯化灭活的PRRSV抗原诱导机体产生的抗主要结构蛋白的抗体滴度较低,说明PRRSV抗原的免疫原性较弱,不能诱导机体产生有效的保护抗体。     

Identification of chitin as a structural component of Giardia cysts.

The intestinal parasite Giardia lamblia is a significant cause of diarrheal disease, which is perpetuated by the infective cyst form of the parasite. Although a rational approach to the control of giardiasis would be to inhibit cyst formation, nothing is known of the chemical composition of the cyst wall or of its biosynthesis. In these studies, we have shown that chitin is a major structural component of G. lamblia and G. muris cyst walls. This conclusion is based on the finding that chitinase specifically destroys the cyst wall, as revealed by electron microscopy. The presence of chitin was also shown directly by lectin binding studies. Of 12 lectins with diverse carbohydrate recognition specificity, only the N-acetylglucosamine-specific lectins wheat germ agglutinin, succinylated wheat germ agglutinin, and tomato lectin bound to cyst walls, as shown by fluorescence microscopy and cytochemistry. Wheat germ agglutinin binding was completely abolished by treatment of the cysts with purified chitinase. This effect was specific since it could be prevented by incubating the enzyme with chitin before treatment of the cysts. Treatment of cysts with N-acetyl-beta-glucosaminidase partially inhibited wheat germ agglutinin binding, whereas other glycosidases and proteases had no effect. These findings indicate that chitin is a major structural component of Giardia cyst walls and raise the possibility that inhibitors of chitin synthesis may be of use in preventing encystation and thus controlling spread of the disease.     

Rubella virus nonstructural protein 2 is a minor immunogen

The full-length nonstructural protein P90 of rubella virus (RV) was expressed as recombinant protein in Escherichia coli bacteria, as well as in Vero cells. Monoclonal antibodies raised against the protein specifically reacted with the protein in both P90-transfected and RV infected Vero cells. Ninety human sera obtained from reconvalescents, vaccinees and patients with acute RV infection were tested for reactivity against the P90 protein. A weak immune reaction was detected only in a small minority (8%), indicating that P90 is minor immunogen for RV and is not suitable for diagnostic purposes.     

Ganglioside GT1b as a complementary receptor component forClostridium botulinumneurotoxins

Clostridium botulinumtype B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.     

The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions

The paramyxovirus simian virus 5 (SV5) cysteine-rich V protein has been shown to be a virus structural protein by analysis of the polypeptides of purified SV5 virions. In addition, the V protein has been identified as a component of the virus nucleocapsid core both by the analysis of the polypeptides present in radioactively labeled preparations of purified nucleocapsids and by immunoelectron microscopy. Quantitative autoradiography was used to determine that there are approximately 350 molecules of the V protein in virions. The V protein has been purified from V recombinant baculovirus-infected insect cells and by using inductively coupled argon plasma atomic emission spectroscopy it was found that each molecule of V binds two zinc atoms.     

A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen.

Cell surface-exposed antigenic determinants of several high-molecular-weight outer membrane proteins of Haemophilus influenzae type b (Hib) have been shown to be consistently immunogenic in human infants convalescing from Hib meningitis. A monoclonal antibody (mab), 6G12, directed against one of these cell surface-exposed outer membrane proteins that has an apparent molecular weight of 98,000 (98K) was identified by radioimmunoprecipitation analysis. Of 120 clinical isolates of Hib, 83 were found to possess antigenic determinants which reacted with mab 6G12 in a colony blot-radioimmunoassay procedure, indicating that the antigenic determinant recognized by mab 6G12 is present in the majority of Hib strains. A different radioimmunoassay, which uses whole Hib cells as antigen, confirmed that strains reactive with mab 6G12 in the colony blot-radioimmunoassay procedure possessed a cell surface-exposed and antibody-accessible antigenic determinant recognized by this mab. Hib strains which did not react with mab 6G12 were found to lack a 98K protein. Passive immunization with mab 6G12 reduced the level of bacteremia that developed in infant rats challenged with the homologous Hib strain against which this mab was raised. In contrast, no protection was observed when the challenge strain was one which lacks the antigenic determinant recognized by mab 6G12. Radioimmunoprecipitation analysis of sera from human infants convalescing from Hib meningitis detected an antibody response directed against the 98K protein. The protection against experimental Hib disease provided by antibody to the 98K protein, the immunogenicity of this protein in human infants, and its presence in a majority of Hib strains indicate that the 98K outer membrane protein may have potential for vaccine development.     

The ninth component of guinea-pig complement. Isolation and identification as an α2-globulin

A method was described for isolation of the ninth component of guinea-pig complement (C9). Injection of the isolated C9 into rabbits resulted in the production of antisera which contained antibody to a serum protein with an electrophoretic mobility of an α₂-globulin. The identity of C9 with the α₂-globulin was proved by means of immunolysoelectrophoresis and quantitative precipitin reaction. The development of the haemolytic band, observed in the α₂-region by overlaying blood agar containing EAC14235678 after immunoelectrophoresis of guinea-pig serum or purified C9, was completely inhibited by the precipitin line formed with IgG fraction of the antisera. Further, upon incubation of the IgG fraction with varying amounts of guinea-pig serum in the presence of ethylene-diaminetetraacetate, more than 99.5 per cent of C9 activity in serum was precipitated in the antibody excess region and the equivalence zone. It was found only in the antigen excess region and all the other eight components remained unaffected in the supernatant fluids.     

Enzyme linked immunosorbent assay for Heymann''s antigen as a contaminating minor component in nephritogenic glycopeptide, nephritogenoside

An enzyme linked immunosorbent assay (ELISA) was introduced for the detection of nephritogenic glycopeptide, nephritogenoside and Heymann's antigen. In the fraction of crude nephritogenoside which induced membranous glomerulonephritis (besides proliferative glomerulonephritis) in rats, both nephritogenoside and Heymann's antigen were detected by ELISA. On the other hand, in the sample of pure nephritogenoside which induced only proliferative glomerulonephritis, Heymann's antigen was not detected at all. These results indicate that crude nephritogenoside preparation contains Heymann's antigen as an inducing factor of membranous glomerulonephritis in homologous animals. In addition to TCA treatment or DEAE-cellulose column chromatography, gel filtration on Bio-Gel P200 was a good tool for the removal of Heymann's antigen from nephritogenoside.     

Identification of potyviral amorphous inclusion protein as a nonstructural, virus-specific protein related to helper component

Antisera to amorphous inclusion (AI) proteins associated with infections by pepper mottle virus (PeMV) and the watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W) were used to probe in vitro translation products of the viral RNAs. The major translation product of PeMV RNA in the rabbit reticulocyte lysate (RRL) system was a previously reported polypeptide of apparent molecular weight 78,000 (Mr 78K). It reacted with anti-AI serum, whereas the major translation product in the wheat germ (WG) system was a 30K polypeptide that did not react with the antiserum. These results, the Mr values, and analyses of peptides generated by partial digestion with proteinase indicate that the amino acid sequences of the 30K polypeptide and the (Mr) 51K AI protein are distinct subsets of the 78K polypeptide amino acid sequence. Similar results were obtained with PRSV-W except that the Mr values of the corresponding translation products are 110K (RRL) and 60K (WG). Thus the 5'-most region of the PeMV and PRSV-W RNAs (corresponding to 78K and 110K, respectively) appears to encode two proteins rather than one as previously supposed on the basis of RRL translation products. Reciprocal serological tests revealed that the tobacco vein mottling virus aphid transmission helper component protein was related to AI protein. There is direct evidence that the AI represent another potyviral-coded nonstructural protein and the first evidence that a biologically functional protein is related to a component of a potyviral inclusion.     

Genetically modified Tomato aspermy virus 2b protein as a tumor-targeting siRNA delivery carrier

In nature, there exist a wide range of dsRNA-binding proteins that have different binding modes for small interfering RNA (siRNA) as well as structural differences, and some of these proteins have potential as effective siRNA delivery carriers. In order to deliver siRNA into cancer cells, a dsRNA-binding 2b protein derived from Tomato aspermy virus was genetically modified by fusing the integrin-targeting RGD peptide to its C-terminus, and biosynthesized. The resulting 2b-RGD protein possesses distinct characteristics favorable for biomedical applications of siRNA: (i) high affinity for siRNA, (ii) siRNA protection against RNases in serum, (iii) low cytotoxicity compared to the polycationic polymers often employed in conventional siRNA carriers, (iv) specific binding to integrins on cancer cells, and the ability to pass through the cell membrane via endocytosis, and (v) the ability to facilitate cytosolic release of siRNA. Here, we demonstrate that the 2b-RGD/siRNA complexes have great potential as a tumor-targeting siRNA delivery carrier and suggest their possible therapeutic applications for cancer treatment.     

Beyond a structural component: sphingolipids in immunology

Two major classes of lipids participating in signaling cascades in immune cells are known today. One comprises glycerol-based lipids with diacylglycerol as its most prominent member that mediates the activation of classical and novel protein kinase C molecules. The second group contains the sphingolipids, with the best-investigated representatives being sphingosine, sphingosine-1-phosphate, and ceramide. In the last years the latter two molecules have especially received considerable attention for their modulatory capacity in the course of an apoptotic response. Today it is clear that sphingolipids are ubiquitously distributed in all eukaryotic cells, especially in cellular membranes, where they were previously thought to fulfil an exclusively structural role. Recent findings, however, have demonstrated functions beyond this. Sphingolipid specific G-protein coupled receptors were identified and their role as intracellular second messengers has been further elucidated. In addition, glycosphingolipids, in particular, are enriched in certain membrane compartments, known as detergent resistant membranes. These serve as entry sites for several receptor-mediated signaling events by stabilizing receptor/kinase interactions, suggesting an involvement in the initiation of signaling cascades. Altogether, these findings have led to new insights into both the role of these lipids in signaling as well as the underlying pathology of several diseases with imbalances in the sphingolipid metabolism. The development of these disorders has mainly been attributed to the toxic potential of lysosphingolipids up to now. In addition, attempts have been made to develop compounds and drugs containing the sphingolipid backbone for influencing diseases associated with unwanted cell activation (e.g, cancer, inflammatory processes). These novel findings and developments are reviewed in the following.     

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2.     

β-Amyloid precursor protein (βAPP) as a marker for axonal injury after head injury

It has been demonstrated recently that β-amyloid protein (βAP), generally associated with the plaques of Alzheimer's disease, can also be found in the brains of survivors of head injury. In this study the distribution of the βAP precursor protein (βAPP) was examined immunohistochemically to determine if it is colocalized with βAP in such cases. βAPP immunoreactivity was observed in neuronal perikarya in the neocortex and in dystrophic neurites surrounding βAP immunoreactive plaques i.e. in a distribution similar to that seen in Alzheimer's disease. In addition, βAPP immunoreactivity was noted within white matter tracts where it marked damaged axons. However, no colocalisation of βAPP with βAP was observed in any white matter region. These results indicate that processing of βAPP to produce βAP occurs in the synaptic terminal field of axons and illustrate the utility of βAPP immunoreactivity as a general marker for axonal injury.