Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit anti-mouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the sections are covered by methyl cellulose and exposed to paraformaldehyde vapour at 80 degrees C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) alpha- and beta-tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

Summary The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit antimouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the paraformaldehyde vapour at 80° C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) - and -tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.Presented in part at the 9th European Congress on Electron Microscopy, York, England, September 4–9, 1988     

Hepatitis B virus core interacts with the host cell nucleolar protein,nucleophosmin 1

Hepatitis B virus (HBV) genome replication requires the packaging of viral factors (pregenomic RNA and polymerase) as well as host factors, including heat shock proteins and protein kinase C. Previous reports have suggested that there are several unidentified host factors that affect this encapsidation step. In this study, we identified a new host factor, nucleophosmin (B23) that interacts with the HBV core protein 149 (Cpl49). We analyzed this factor using NHS-activated sepharose resin and MALDI-TOF MS. Using the BIAcore analysis system, we were also able to deduce that the B23.1 residues 259–294 were required for the interaction between Cpl49 and B23.1 in vitro.     

Interaction of viral proteins with host cell death machinery

In recent years, intense research has been directed towards understanding molecular mechanisms involved in viral pathogenesis. It is now known that many viruses manipulate host defense mechanisms to prevent apoptosis in order to maximize viral replication. Towards the end of their replication cycle, certain viruses direct the synthesis of proteins that induce apoptosis or cell lysis thereby facilitating viral release from the cell. The present review summarizes the current understanding of interactions between viral proteins and the host cell death machinery.     

弓形虫与宿主细胞相互作用研究进展

刚地弓形虫(Toxoplasma gondii)在细胞内严格寄生,因此它能引起哺乳类宿主(包括人类)细胞的感染。凋亡在宿主细胞与弓形虫的相互作用中发挥着重要的作用。在未受感染的宿主细胞中,凋亡被间接机制所限制,因而宿主细胞能够对弓形虫发生炎症反应。与之相反,在被感染的宿主细胞中,由于凋亡信号级联反应直接受到了干扰,从而抑制了宿主细胞凋亡,这就有利于弓形虫在宿主细胞内的生存和发育。值得注意的是,弓形虫调节和抑制凋亡的两种能力,需要一个精密的调节系统来调控弓形虫和宿主细胞的相互作用,以维持弓形虫稳定的持续感染。重点从弓形虫有关的宿主细胞的凋亡方面进行了介绍。     

The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus

The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.     

Studies on the human tumor nucleolar antigens

With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat antirabbit antibodies, bright nucleolar immunofluorescence was observed in human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in most normal tissue specimens, benign adenomas, hyperplastic tissues, and specimens of inflammatory diseases. A study was made on the presence in benign and malignant breast tumors of a common nucleolar antigen previously found in a broad range of human malignant tumors. Bright nucleolar immunofluorescence was observed in 19/20 (95%) of known breast cancer specimens. In the group of 80 unknown samples in the "blind" study, 75 (94%) were correctly identified as malignant or benign on the basis of the presence and distribution of the nucleolar fluorescence. In a group of 67 samples in which the nucleolar fluorescence was either readily observed or virtually absent, 47/48 (98%) of the malignant tumors were correctly identified. Of the bening lesions or normal breast specimens, 18/19 (95%) were correctly identified as negative for nucleolar fluorescence. These studies extend the results previously reported for a common nucleolar antigen in a broad range of human cancers to a larger series of malignancies of a particular organ. The tumor nucleolar antigen(s) were partially characterized by isoelectric focusing on 4% polyacrylamide gels. One major band had a pI of 6.3 and a minor band had a pI of 6.1. These antigens were not found in the normal human liver nucleoli.     

Interaction of hepatitis C virus proteins with host cell membranes and lipids

For replication, viruses depend on specific components and energy supplies from the host cell. The main steps in the lifecycle of positive-strand RNA viruses depend on cellular membranes. Interest is increasing in studying the interactions between host cell membranes and viral proteins to understand how such viruses replicate their genome and produce infectious particles. These studies should also lead to a better knowledge of the different mechanisms underlying membrane-protein associations. The various molecular interactions of hepatitis C virus proteins with the membranes and lipids of the infected cell highlight how a virus can exploit the diversity of interactions that occur between proteins and membranes or lipid structures.     

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.     

Interaction of Leishmania with the host macrophage

The leishmaniases are a group of diseases with a spectrum of clinical manifestations ranging from self-healing cutaneous ulcers to severe visceral disease and even death. In mammals, the macrophage is the main host for the Leishmania amastigote. However, the macrophage is also the immune effector cell that, upon activation, is able to kill intracellular organisms. Therefore, understanding the parasite mechanisms which allow establishment of infection, and the host immune mechanisms that are responsible for parasite recognition and killing should lead to the development of new drugs and vaccines.     

Interaction of nucleolar phosphoprotein B23 with nucleic acids

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.     

Cellular distribution of the hamster liver specific nucleolar antigens

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.     

Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex.

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.