Promotion of virus assembly and organization by the measles virus matrix protein.

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.

A residue located at the junction of the head and stalk regions of measles virus fusion protein regulates membrane fusion by controlling conformational stability.

The fusion (F) protein of measles virus performs refolding from the thermodynamically metastable prefusion form to the highly stable postfusion form via an activated unstable intermediate stage, to induce membrane fusion. Some amino acids involved in the fusion regulation cluster in the heptad repeat B (HR-B) domain of the stalk region, among which substitution of residue 465 by various amino acids revealed that fusion activity correlates well with its side chain length from the Cα (P<0.01) and van der Waals volume (P<0.001), except for Phe, Tyr, Trp, Pro and His carrying ring structures. Directed towards the head region, longer side chains of the non-ring-type 465 residues penetrate more deeply into the head region and may disturb the hydrophobic interaction between the stalk and head regions and cause destabilization of the molecule by lowering the energy barrier for refolding, which conferred the F protein enhanced fusion activity. Contrarily, the side chain of ring-type 465 residues turned away from the head region, resulting in not only no contact with the head region but also extensive coverage of the HR-B surface, which may prevent the dissociation of the HR-B bundle for initiation of membrane fusion and suppress fusion activity. Located in the HR-B domain just at the junction between the head and stalk regions, amino acid 465 is endowed with a possible ability to either destabilize or stabilize the F protein depending on its molecular volume and the direction of the side chain, regulating fusion activity of measles virus F protein.

Histologic otosclerosis is associated with the presence of measles virus in the stapes footplate.

HYPOTHESIS: Persistent measles virus infection of the otic capsule is presumed to be one of the etiologic factors in otosclerosis. The viral pathogenesis of otosclerosis could be established only by correlative analysis: histologic examination of the stapes footplates and reverse-transcriptase polymerase chain reaction amplification of the viral RNA. At present, histologic analysis of the removed stapes footplates is the only appropriate method of distinguishing otosclerotic and nonotosclerotic stapes fixations. BACKGROUND: The presence of measles virus was shown in otosclerotic patients by reverse-transcriptase polymerase chain reaction amplification of the viral RNA and detecting the viral proteins by immunohistochemistry. METHODS: Nucleic acids (mRNA, vRNA, and DNA) were extracted from ankylotic stapes footplates of stapes fixation patients (n = 44). Measles virus genomic nucleoprotein RNA was amplified by seminested reverse-transcriptase polymerase chain reaction. Amplification results were correlated to postoperative histologic findings. RESULTS: Measles virus RNA was detectable only in histologically otosclerotic stapes footplates (n = 32). Histology for virus-negative footplates (n = 12) excluded otosclerosis. Virus-negative stapes footplates showed annular calcification (n = 8), bone resorption with increased numbers of hemosiderophages (n = 2), and mononuclear cell infiltration with osteolysis (n = 2). CONCLUSION: Stapes ankylosis is a heterogenous disease causing conductive hearing loss with different causes. Nonotosclerotic stapes fixations may belong to degenerative disorders with variable histopathology. Otosclerosis is an inflammatory disease resulting from persisting measles virus infection of the otic capsule.

CD46-mediated measles virus entry: a first key to host-range specificity.

Humans are the sole natural host of measles virus. The identification of CD46 as a virus receptor and of the involvement of moesin sheds some light on the molecular events occurring during virus entry into the cell. Knowledge of the key role of CD46 paves the way to creating transgenic mice sensitive to measles virus infection.

Persistent measles virus infection of the intestine: confirmation by immunogold electron microscopy.

This study sought to investigate persistent measles virus infection of the intestine: a novel protocol for immunogold electron microscopy was developed using a polyclonal anti-measles nucleoprotein antibody on reprocessed, formalin fixed paraffin wax embedded tissue sections. Antibody binding was detected using both immunoperoxidase and light microscopy on tissue sections, and 10 nm gold conjugated secondary antibody and electron microscopy on ultrathin sections. The techniques were validated using both measles infected vero cells and human tissues with established measles infection: these included brain affected by subacute sclerosing panencephalitis and acute measles appendicitis. The technique was applied subsequently to six untreated cases of granulomatous Crohn's disease, and two cases of ileocaecal tuberculosis, a granulomatous control. Mumps primary antibody--applied to both mumps infected vero cells, and measles infected vero cells and tissues studied by immunoperoxidase, and measles antibody on mumps infected cells studied by immunoperoxidase and immunogold--were used as specificity controls: the primary antibodies identified their respective target antigen and there was no antibody cross reactivity. Measles virus nucleocapsids labelled with gold conjugated antibody in both infected cells and tissues, including foci of granulomatous inflammation in five of six cases of Crohn's disease: in the fifth case, the granuloma could not be identified in ultrathin section. In one of the tuberculosis cases, a low level of signal was noted while the second case was negative. Labelling adopted a characteristic pattern in all infected tissues, strengthening the specificity of these findings. This study provides the first direct confirmation of persistent measles virus infection of the intestine.

Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures.

The gene encoding the major nucleocapsid, N, protein of measles virus has been inserted into a baculovirus vector under the control of the polyhedrin promoter. Insect cells infected with this recombinant baculovirus synthesize high levels of measles N protein, up to 40% of total soluble cell protein. The recombinant protein is recognized by sera from convalescent patients, vaccinees and patients with subacute sclerosing panencephalitis and thus could form the basis of a simple diagnostic assay. Nucleocapsid-like structures, similar to those found in mammalian cells infected with measles virus, can be observed in both the nucleus and cytoplasm of the infected insect cells. These have many structural features in common with nucleocapsids found in measles virus-infected cells, but are longer (up to 2 microns) and have a lower buoyant density. Measles N protein thus appears to be capable of assembling into nucleocapsid-like structures in the absence of measles virion RNA or other viral proteins.

Conformational maturation of measles virus nucleocapsid protein.

We have obtained a polyclonal antiserum, N-BE, against the denatured, amino-terminal half of the measles virus (MV) nucleocapsid (N) protein and a monoclonal antibody (MAb), N46, which recognizes a conformation-dependent epitope in the same region. Amino acid residues 23 to 239 were required and sufficient for the formation of the conformational epitope. Using these antibodies, we show that the N protein of MV is synthesized as a relatively unfolded protein which first appears in the free-protein pool. This nascent N protein undergoes a conformational change into a more folded mature form. This change does not require the participation of other viral proteins or genomic RNA. The mature N protein does not accumulate in the free-protein pool but is quickly and selectively incorporated into the viral nucleocapsids. The mature N protein is a target for interaction with the phosphoprotein (P protein) of MV. This interaction interferes with the recognition of the N protein by the N46 MAb. This suggests that the association with the P protein may mask the binding site for the N46 MAb or that it induces a conformational change in the N protein.

Label-fracture of plasma membranes isolated from measles virus-infected cells.

We present a method that permits correlation of the intramembrane architecture of plasma membrane fracture faces with the distribution of specific molecules at the corresponding cytoplasmic or exoplasmic membrane surfaces. HeLa cells infected with measles virus were used as a model system. Large fragments of the dorsal membrane were isolated after the virus glycoproteins were tagged at the outer cell surface with immune serum and protein A-gold markers. In a second step, different virus polypeptides at the inner cell surface were also identified by a smaller gold label. Thereafter, the isolated plasma membranes were frozen and freeze-fractured. The complementary fracture faces were shadowed with heavy metals and carbon and examined in the transmission electron microscope without cleaning of remaining biological material. Thus, the micromorphology of the replicated fracture faces and the topochemistry of virus components localized at the corresponding leaflets of the plasmalemma could be seen on the same image at high resolution. Of note is that the freeze-fracture morphology of the protoplasmic face is related to the molecular composition of the cytoplasmic surface, as revealed by antibody tagging.

Evidence of persistent measles virus infection in Crohn's disease.

Transmission electron microscopy was used to examine the microvasculature of perfusion-fixed tissues from Crohn's disease and control patients. Paramyxovirus-like particles, and inclusions consisting of condensations of nucleocapsid, in giant cells and endothelium at foci of vascular injury were identified in all 9 Crohn's disease patients. Tissues from patients with Crohn's disease were also examined by either in situ hybridisation (n = 10) or immunohistochemistry (n = 15), and compared to inflammatory and noninflammatory controls (n = 22). Hybridisation for measles virus N-protein genomic RNA was positive in all cases of Crohn's disease localising to foci of granulomatous vasculitis and lymphoid follicles. Positive immunohistochemical staining for measles virus nucleocapsid protein was positive in 13 of 15 patients with Crohn's disease, localising to foci of granulomatous inflammation. Hybridisation for measles virus RNA was positive in a minority of control intestinal tissues; viral inclusions were not seen ultrastructurally. Immunostaining was negative in control cases of intestinal tuberculosis. These observations suggest that measles virus is capable of causing persistent infection of the intestine and that Crohn's disease may be caused by a granulomatous vasculitis in response to this virus.

In situ analysis of Paget's disease of bone for measles-specific PCR-amplified cDNA.

Paget's disease of bone is a disease of unknown etiology. The demonstration of viral-like particles on ultrastructural examination and the putative detection of viral antibodies and nucleic acids in the tissues suggest a possible viral association. The purpose of this study was to search for nucleic acid sequences homologous to measles virus using the recently described reverse transcriptase (RT) polymerase chain reaction (PCR) in situ hybridization (ISH) technique. After performing RT PCR ISH utilizing primers specific for the nucleocapsid region of the measles virus, an intense signal was evident in most measles-infected HeLa cells compared with a weak signal in few of these cells using standard cDNA-RNA ISH analysis. Amplified measles nucleic acid was detected in tissue from a patient who died of measles infection and was not detected in any of the 11 cases of Paget's disease of bone studied or in a giant cell tumor of bone that had tubuloreticular inclusions on electron microscopy. Therefore, these data suggest that infection by the measles virus is not associated with Paget's disease of bone.

Measles virus from a long-term persistently infected human T lymphoblastoid cell line, in contrast to the cytocidal parental virus, establishes an immediate persistence in the original cell line.

To investigate the mechanisms of measles virus (MV) establishment and maintenance of persistence in lymphoid cells, we have established a long-term persistent infection with MV, Edmonston strain, in the human T lymphoblastoid cell line MOLT4, which has been in continuous culture for over 8 years. In this culture, designated MOMP1, more than 98% of cells display viral antigens. The MOMP1 culture is immune to superinfection with MV and is not cured by anti-MV antibodies. No evidence of defective interfering particles was obtained. The persistently infected culture releases an infectious virus showing a miniplaque and thermoresistant modified phenotype that, unlike the parental virus Edmonston strain which produces a lytic infection with extensive cell fusion, establishes an immediate persistence in MOLT4 cells with neither significant loss of cell viability nor cell fusion. This suggests that the modification in the virus suffices to maintain the state of persistence without requiring a coevolution of the host cell during the infection, as has been reported in other persistent virus infections.

Acute measles encephalitis of the delayed type: neuroradiological and immunological findings.

A case of delayed acute measles encephalitis in an immunosuppressed child is reported. Detailed immunological studies have shown defective humoral immunity (defective IgA, IgG2 and IgG3) and decreased natural killer activity. Neuroradiological examination by magnetic resonance imaging revealed several high signal lesions on T2-weighted images in the gray matter without clinical or pathological correlation. The implications of these findings are discussed.

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.

[The isolation of the morbilli virus from the Baikal seal Phoca sibirica and its preliminary characterization]. / Vydelenie morbillivirusa ot baikal'skoi nerpy Phoca sibirica i ego predvaritel'naia kharakteristika.

A morbillivirus was isolated from the organs of a seal (Phoca sibirica) which had died during 1987-1988 epizootic in Baikal. This Baikal seal morbillivirus (BSM) was adapted to Vero cell cultures in which it induced a cytopathic effect developing to complete destruction of the monolayer. Typing of BSM was done by indirect immunofluorescence test and enzyme immunoassay using antibodies to distemper and measles viruses. A method for virus concentration and purification was developed. According to electron microscopic examinations, the virus virions were spherical particles of heterogeneous sizes over 100 nm in diameter. The clinical picture of seal infection, pathological anatomy and histopathology are described. A possible role of BSM in the epizootics of Baikal seals is discussed.

Immunoelectron microscopical labelling of a glycolipid in the envelopes of brain cell-derived budding viruses, Semliki Forest, influenza and measles, using a monoclonal antibody directed chiefly against galactocerebroside resulting from Semliki Forest virus infection.

Neurotropic RNA budding viruses such as Semliki Forest virus (SFV), influenza and measles were each grown in identical mouse brain cell cultures. Positive immunoelectron microscopical labelling with gold was seen in the envelope of these viruses using an anti-SFV derived glycolipid monoclonal antibody (MAb), 373 shown to be directed chiefly against galactocerebroside. The results indicate that each enveloped virus grown from the same cell type contains the same glycolipid in its envelope. The presence of common glycolipids derived from the host cell in the envelopes of various enveloped budding viruses may play a significant role in the pathogenesis of virus induced, immune mediated CNS autoimmunity and demyelination, particularly in multiple sclerosis (MS).

Cytopathic effects of measles virus on the human intestinal mucosa.

We reported a case of measles with diarrhea in an infant. Capsule biopsy samples of intestinal mucosa taken 14 days after onset of fever was damaged and had low disaccharidase activities. Virus particles were found in the damaged villous cell. Second intestinal mucosa biopsy samples taken 28 days after onset of fever was found to be normal in structure, but lactase and sucrase activities were still low. Mucosal damage by virus particles was the cause of diarrhea in our infant.

[Electron microscopy study of the nucleocapsid structure of the measles virus isolated from cell cultures with primary and chronic infections]. / Elektronno-mikroskopicheskoe izuchenie struktury nukleokapsidov virusa kori, vydelennykh iz kul'tur kletok pri pervichnoi i khronicheskoi infektsii.

Electron microscopic examination of isolated intracellular measles virus nucleocapsids (NC) revealed a relationship between their structure, cell system, and the type of infection. Acute virus infection of Vero or Japanese quail embryo cells gave rise to the formation of linear NC strands with regularly and tightly stacked turns. Acutely infected L-41 or HEp-2 cells contained heteromorphous viral NC populations which included both typical and loosely packed NC. Persistently infected L-41 and Hep-2 cells predominantly contained NC of the latter type with the appearance of a "strings of beads".

Replica-immunogold technique applied to studies on measles virus morphogenesis.

The replica technique was applied to studies on the dynamic process of measles virus budding on infected HeLa cells. Virus structures were identified by labeling with anti-measles antibodies and protein A-gold. The combination of these two methods enabled us to characterize the sequence of virus budding at the plasma membrane, to localize virus structures on cytoskeletons of infected cells, and to study the influence of Ca2+ ions on virus structures at the plasma membrane. Studies on platinum carbon surface replicas suggest that the process of virus budding is similar to the genesis of cellular microvilli. Replicas prepared from cytoskeletons of infected cells reveal a close association of budding virus with actin filaments composing the outer parts of the networks. Replicas of apical plasma membranes isolated from infected cells show the attachment of viral nucleocapsids to the protoplasmic membrane face of infected cells. These nucleocapsids are not present on membranes prepared from cells treated with calcium and the ionophore A23187. In addition viral cell surface antigens become randomly distributed on these cells. The data suggest that measles virus morphogenesis at the plasma membrane of cultured cells is dependent on the function of the cytoskeleton and may be influenced by Ca2+ ions.

Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells.

Cytoskeletons were prepared from measles virus infected HeLa cells to investigate the involvement of cytoskeletal filaments in virus budding at the plasma membrane. The cytoskeletons retained nearly 80% of measles virus hemagglutinin, the major viral polypeptides, including P, NP, and M, and 2 to 12% of the total cell bound infectivity. As demonstrated with platinum- and carbon-shadowed cytoskeletons, all stages of budding, i.e., virus specific strands, stub-like protrusions, and completely rounded virus particles, are associated with actin filaments composing the outer part of the cytoskeletal network. As shown with ultrathin sections of flat embedded extracted cells, actin filaments identified with heavy meromyosin almost exclusively protrude into virus particles with their barbed ends and are in close association with viral nucleocapsids. The data support previous suggestions that actin is involved in virus budding and show that budding itself is possibly the result of a vectorial growth of actin filaments.