Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigen-induced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Nucleotide sequence and host La protein interactions of rabies virus leader RNA.

Rabies virus leader RNA was detected in infected BHK-21 cell extracts by hybridization to end-labeled genomic RNA. Similar to the leader RNA of vesicular stomatitis virus, the leader RNA of rabies virus was also found to be associated with the La protein by specific immunoprecipitation with antisera from lupus patients. The 3' end of the genomic RNA of rabies virus was sequenced, and the size and termination site of leader RNA were determined. In addition, extension of the sequence into the nucleocapsid gene of rabies virus showed an open reading frame for at least 37 amino acid residues. Sequence relationships between rabies virus and vesicular stomatitis virus leader genes and the possible involvement of the La protein in rhabdovirus biology are discussed.     

Studies on the Nucleocapsid Structure of a Group A Arbovirus

When Sindbis virus (273S) was treated with sodium desoxycholate, a nonhemagglutinating 136S particle was liberated from the virion, representing the viral nucleocapsid (core). Electron microscopically it appeared as a spherical particle 35 nm in diameter, showing ringlike morphological units 12 to 14 nm in diameter on its surface. When the one- and two-sided images of core particles were correlated, their structure could be demonstrated to have the T = 3 arrangement of 32 hexamer-pentamer morphological units within a symmetrical surface lattice. The core contained a further spherical structure (12 to 16 nm in diameter) which was designated as the central core component. Two proteins were found associated with the core, a third viral protein belonged to the hemagglutinating surface structures. The significance of these findings for virus classification is discussed.     

Quaternary structure of the immunostimulating complex (Iscom)

Proteins of either HIV-1, hepatitis B, or rabies virus were incorporated with the adjuvant substance Quil A and cholesterol into the immunostimulating complex: iscom. Formation and symmetry of this regular complex were analyzed by electron microscopy. Micellar structures with a diameter of about 12 nm, occasionally with a 7-nm stain-filled center, were formed in a 0.03% water suspension of Quil A. Cavities or holes appeared in the smooth structures of cholesterol upon the addition ofing Quil A, and after mixing Quil A and cholesterol 1:1 fragile and flattened structures of matrix were produced withth a diameter of about 40 nm. By freeze-drying the matrix was preserved as a cage-like, isometric particle. Stable iscom particles composed of Quil A, cholesterol, and selected viral proteins had an approximate diameter of 32 nm. The particles had an uniform, cage-like structure, exhibiting icosahedral symmetry, irrespective of the viral proteins incorporated. Tilting experiments and rotational image analysis indicated that the iscoms were composed of 20 morphological subunits assembled in a pentagonal dodecahedron with a hole on each of the 12 pentagonal faces. The symmetrical shape of the iscom might explain both its remarkable stability and its capacity to efficiently present antigens to the immune system.     

Helical Nucleocapsid Structure of the Oncogenic Ribonucleic Acid Viruses (Oncornaviruses)

Negative staining of virions and isolated nucleoids from avian myeloblastosis virus, murine leukemia virus, murine mammary tumor virus, and feline leukemia virus reveals common internal structures. The majority of virions that are penetrated by phosphotungstate show spherical nucleoids with no apparent symmetry. In a small percentage of virions, two distinctive structures are found: (i) single strands (3 to 5 nm in diameter) which are presumed to be the nucleoprotein and are found randomly oriented throughout the viral interior and (ii) helical structures (7 to 9 nm in diameter) which contain these nucleoprotein strands and are observed at the periphery of the nucleoid. The finding of helical nucleocapsid segments at the periphery of the nucleoid, as well as the hollow spherical structure observed in thin section of budding virions, has led to the hypothesis that the nucleocapsid of the freshly budded oncornavirus is supercoiled as a hollow sphere. This symmetry, however, is considered transient, as the internal structure of the extracellular virus undergoes a conformational rearrangement; thus, due to structural instability, the nucleocapsid uncoils and the nucleoprotein strands fill the interior of the virion. The extracellular virion is therefore considered degenerate in respect to symmetry, explaining the difficulty in detecting a helical nucleocapsid.     

Fine Structure and Morphogenesis of Borna Disease Virus

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.     

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus, murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measles virus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient as with infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable by antibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can be presented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsid proteins contribute to class II-restricted measles virus-specific CTL responses. These results also show that endogenously synthesized measles virus proteins can be efficiently presented by class II antigens. The implications of these findings for measles virus pathogenesis and for antigen processing are discussed.     

鸭肠炎病毒CHv强毒株超微结构研究

将鸭成纤维细胞培养的鸭肠炎病毒(DEV),经超声处理、高速冷冻差速离心后,采用酒石酸钾—甘油非线性密度梯度超速离心,收集病毒蛋白带,3%磷钨酸负染后观察病毒粒子形态。结果表明:病毒粒子主要集中在40%~50%酒石酸钾—甘油缓冲液交界层。电镜下,病毒粒子纯净,具有疱疹病毒典型形态结构,剖面六角,外观轮廓清楚。成熟病毒粒子直径约150~266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构。多数病毒粒子以单核衣壳为主,一定数量的病毒具有双核衣壳,偶见三核衣壳,核衣壳直径为100~150nm,呈现致密圆形、半圆形或马蹄形等类型。在核衣壳外和囊膜之间可见明显的亮晕。核心DNA电子染色较深,集中分布,直径40~65nm。本文获得的清晰DEV负染超微结构照片,为该病毒结构生物学的研究提供了重要依据。     

Filamentous structures associated with Epstein-Barr virus-infected cells.

After the onset of Epstein-Barr virus DNA and protein synthesis 10 h after superinfection of Raji cells (a cell line containing Epstein-Barr virus DNA but not producing virus), filamentous structures 25 nm in diameter and 0.2 to 1.4 micrometers in length could be detected in the cell cytoplasm by electron microscopy. These structures banded in metrizamide gradients with viral DNA and proteins, but at a density different from that of virions or nucleocapsids. These filaments, enriched in a 155,000-dalton protein similar in size to a major nucleocapsid protein of Epstein-Barr virus, may represent intermediates in viral nucleocapsid assembly.     

家蚕核型多角体病毒及核衣壳的纯化和其结构蛋白的凝胶电泳分析

 用差速离心和蔗糖密度梯度离心提纯了家蚕核型多角体病毒及其核衣壳,并用SDS聚丙烯酰胺凝胶(均一胶和梯度胶)电泳分析了它们的结构蛋白。测量使用游标卡尺,计算使用微型计算机。凝胶用很染色方法染色。 结果表明,家蚕核型多角体病毒长381.2±17.81纳米,直径88.86±9.605纳米;核衣壳长328.9±5.917纳米,直径41.36±1.167纳米。两种凝胶电泳所得数据显示病毒粒子至少含有20组分子量不同的结构多肽,含量较多的是P31、P40、P28和P20。P45和一些小分子多肽看来主要存于囊膜上。P31的分子量与多角体蛋白相同,我们的实验表明它可能是病毒粒子和核衣壳的结构组分。     

Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site

Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.     

Transmembrane communication in cells chronically infected with measles virus

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.     

The first report on the characterization of a virus isolated from Allomyrina dichotoma in the Republic of Korea

This report reveals the structure of a virus extracted from the Korean horn beetle Allomyrina dichotoma. The purified virus particle was 100% identical to Allomyrina virus lef‐8 sequence registered as KM_233709.1. The structure of this virus was confirmed to be closely related to that of the Nudiviridae family, and it was rod shaped and enveloped, and observed to be of approximately the mean length of a single viral nucleocapsid of 200–210 nm and mean diameter of 100–110 nm. These results provide an insight into the structural characteristics of the Nudiviridae family that can be used for nudiviral identification.     

Morphology and Morphogenesis of Sindbis Virus as Seen with Freeze-Etching Techniques

Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.     

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.