Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis.

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the brain sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.     

Electrophoretic Distribution of the Proteins and Glycoproteins of Influenza Virus and Sendai Virus

The proteins of influenza (WSN) and Sendai virus have been separated by polyacrylamide gel electrophoresis into five components. In both cases, three of these components were shown to be glycoproteins containing fucose, galactose, and glucosamine. Two protein components of each virus were probably free from these sugar residues, including the structural unit of the viral ribonucleoprotein (molecular weight of about 60,000 daltons).     

Structural studies on the Ebola virus matrix protein VP40 indicate that matrix proteins of enveloped RNA viruses are analogues but not homologues

Matrix proteins are the driving force of assembly of enveloped viruses. Their main function is to interact with and polymerize at cellular membranes and link other viral components to the matrix-membrane complex resulting in individual particle shapes and ensuring the integrity of the viral particle. Although matrix proteins of different virus families show functional analogy, they share no sequence or structural homology, Their diversity is also evident in that they use a variety of late domain motifs to commit the cellular vacuolar protein sorting machinery to virus budding. Here, we discuss the structural and functional aspects of teh filovirus matrix protein VP40 and compare them to other known matrix protein structures from vesicular stomatitis virus adn retroviral matrix protein.     

Mucin biopolymers as broad-spectrum antiviral agents

Mucus is a porous biopolymer matrix that coats all wet epithelia in the human body and serves as the first line of defense against many pathogenic bacteria and viruses. However, under certain conditions viruses are able to penetrate this infection barrier, which compromises the protective function of native mucus. Here, we find that isolated porcine gastric mucin polymers, key structural components of native mucus, can protect an underlying cell layer from infection by small viruses such as human papillomavirus (HPV), Merkel cell polyomavirus (MCV), or a strain of influenza A virus. Single particle analysis of virus mobility inside the mucin barrier reveals that this shielding effect is in part based on a retardation of virus diffusion inside the biopolymer matrix. Our findings suggest that purified mucins may be used as a broad-range antiviral supplement to personal hygiene products, baby formula or lubricants to support our immune system.     

Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.     

Structural and Nonstructural Proteins of an Arbovirus

Purified Semliki Forest virus (SFV) contains three structural proteins while its core (nucleocapsid) contains two of these proteins. To identify all of the proteins synthesized under virus direction, cells were infected with SFV in the presence of actinomycin D and guanidine. Cell protein synthesis was markedly and irreversibly inhibited under these conditions; virus growth was reversibly inhibited by guanidine and began when the cells were washed to remove the guanidine. When cells were treated with guanidine for 4 hr after virus infection and then were washed, five major proteins were produced early in infection. Three of these proteins corresponded to virus structural proteins. None of these five proteins was a major protein of uninfected cells or of virus-infected cells which had been incubated with partially purified interferon before infection. Late in infection, three major proteins, the virus structural proteins, were produced.     

The fine structure of the influenza virus envelope and the concept of transmembrane asymmetry of lateral domains in biomembranes

The molecular architectures of enveloped viruses provide a demonstrative example of perfectly arranged macromolecular complexes, which are formed via highly specific interactions of all structural components. Virus morphogenesis is a multistep process that depends on the concerted actions of many viral and cell components, as well as a fitted organization of main viral constituents. The virus envelope is composed of a mixture of lipid raft and nonraft domains. The domains are recruited from the host cell membrane as discrete well-ordered lipid-protein units during virus assembly. The raft-like nature of the influenza virus A envelope was visualized using a novel approach of cold solubilization of detergent-resistant membranes from intact influenza virus A virions with a mixture of NP40 and octyl glucopyranoside, two nonionic detergents drastically differing in their raft-solubilizing activities. The virus envelope is apparently an ensemble of flexibly joint platforms, which are composed of surface glycoproteins (hemagglutinin and neuraminidase), the matrix M1 protein, and lipids. The modern concept of the transmembrane asymmetry of lateral domains in biological membranes was used to explain the solubilization mechanism revealed. Based on the principles of this concept, the M1 protein shell was assumed to provide a structure-forming framework to support asymmetrical rafts in the virus envelope.     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.     

Specific antibodies to viruses HL-23 and BILN in the blood plasma of patients with acute myelogenous leukaemia and with potential preleukaemia

Blood plasma samples from patients with acute myelogenous leukaemia (AML) or potential preleukaemia and from control subjects were tested for antibodies to the viruses HL-23 and BILN by membrane immunofluorescence. Of 15 patients with untreated AML, three, each having a low peripheral leucocyte count at the time of sampling, had detectable antibodies. Antibodies were present in the plasma of 5 out of 8 AML patients being in remission as a result of chemotherapy. In these cases, the antibody levels significantly exceeded those demonstrated in the untreated cases. Of 12 patients with potential preleukaemia, five proved to be positive. Of the 7 antibody-negative patients, four developed manifest leukaemia within 12-18 months after the first testing. The results are suggestive of a favourable prognostic role of the presence of the antibodies under study. In the majority of the antibody-positive AML and potential preleukameia cases antibodies were detectable to both components of the HL-23 virus. Of 30 control subjects, three had demonstrable antibodies to the BILN virus.     

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons.

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.     

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.     

Analysis of structural proteins of measles, canine distemper, and rinderpest viruses

Serological relationships among measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RV), which constitute morbillivirus subgroup of paramyxoviridae, were investigated by immunoprecipitation and SDS-polyacrylamide gel electrophoresis for their major structural proteins, i.e., hemagglutinin (H), nucleocapsid (NC), fusion (F), and matrix (M) proteins. The molecular weights of the four structural proteins of MV and CDV were confirmed to correspond to those previously reported by several investigators. Structural proteins of RV were analyzed for the first time in the present study and found to have molecular weights of 74,000, 62,000, 44,000, and 40,000 for H, HC, F, and M proteins, respectively. By labeling with glucosamine, the presence of carbohydrate moiety was found in H protein for all the three viruses and in F protein of CDV. The serums from the convalescent animals infected with respective virus disclosed one-way cross pattern depending on the combinations of virus and antiserums, but failed to show the reciprocal cross reactivity. On the other hand, hyperimmune serums to respective virus showed the reciprocal cross-reactivity with the four structural proteins indicating that each of the major structural proteins possesses the antigen common to all three morbilliviruses.     

Phosphorylation of Simian Virus 40 Proteins in a Cell-Free System

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.     

Separation and Structure of Components of Nuclear Polyhedrosis Virus of the Silkworm

Morphology of structural components of nuclear polyhedrosis virus (NPV) particles of the silkworm (Bombyx mori Linné) was studied by electron microscope using negative staining. NPV particles isolated from polyhedra could be separated into five structural components by centrifugation in sucrose density gradients. The lowest band (band I) was found to consist of thick rod-shaped particles (330 by 80 nm) with knobby surfaces and with occasional protrusion at one end. The second band from the bottom (band II) was shown to consist mainly of slender rod-shaped particles (360 by 60 nm), in which internal structures were visible as a dense mass. Regular striations were also seen on the surface of these particles. By treatment with mercaptoethanol, these particles were drastically damaged, and in some cases the internal substances were partially released, producing empty inner membranes of various degrees of disintegration. In bands III and IV, both empty spherical and empty rod-shaped membranes were present. Band III was rich in empty spherical membranes which were shown to be the outer membranes of thick rod-shaped particles. The empty rod-shaped membranes, the inner membranes, were mainly located in band IV and have cross striations on the surface. It is remarkable that the uppermost band (band V) consisted purely of small spherical particles, somewhat heterogeneous in size and shape (around 20 to 25 nm in diameter), indicating the particles to be the degradation product of the virus particles. Similar particles could also be observed within the empty inner membranes.     

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.     

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.     

Variation in two components of pathogenicity and its relationship with genetic diversity in Plasmopara halstedii (sunflower downy mildew)

The specificity of the two components of pathogenicity: virulence and aggressiveness and its relationship with genetic variability were analysed in a local Plasmopara halstedii (sunflower downy mildew) population. Pathogenic and molecular analyses were carried out on seven isolates including five progeny isolates of five races arising from two parental races 100 and 710. P. halstedii isolates showed significant differences for all aggressiveness criteria and important genetic variations. Three cases of relationship between virulence and aggressiveness for progeny isolates as compared with parental ones were found as positive, negative or uncorrelated. For solving the specificity of these cases, relationship between the two components of pathogenicity among the isolates of three different races localised in the same genetic clade was positive. The hypothesis explaining these cases is discussed.     

Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study

S. Singh and R. Gupta Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study Objectives: To evaluate the utility of image analysis using textural parameters obtained from a co‐occurrence matrix in differentiating the three components of fibroadenoma of the breast, in fine needle aspirate smears. Methods: Sixty cases of histologically proven fibroadenoma were included in this study. Of these, 40 cases were used as a training set and 20 cases were taken as a test set for the discriminant analysis. Digital images were acquired from cytological preparations of all the cases and three components of fibroadenoma (namely, monolayered cell clusters, stromal fragments and background with bare nuclei) were selected for image analysis. A co‐occurrence matrix was generated and a texture parameter vector (sum mean, energy, entropy, contrast, cluster tendency and homogeneity) was calculated for each pixel. The percentage of pixels correctly classified to a component of fibroadenoma on discriminant analysis was noted. Results: The textural parameters, when considered in isolation, showed considerable overlap in their values of the three cytological components of fibroadenoma. However, the stepwise discriminant analysis revealed that all six textural parameters contributed significantly to the discriminant functions. Discriminant analysis using all the six parameters showed that the numbers of pixels correctly classified in training and tests sets were 96.7% and 93.0%, respectively. Conclusion: Textural analysis using a co‐occurrence matrix appears to be useful in differentiating the three cytological components of fibroadenoma. These results could further be utilized in developing algorithms for image segmentation and automated diagnosis, but need to be confirmed in further studies.     

Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV_239Δnef and SIV_PBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV_239Δnef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIV_PBj6.6Δnef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV_89.6PD, by intravenous injection. All of the SIV_239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIV_PBj6.6Δnef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Disordered regions in C-domain structure of influenza virus M1 protein

Influenza virus matrix M1 protein is one of the main structural components of the virion performing also many different functions in infected cell. X-ray analysis data with 2.08 angstrom resolution were obtained only for the N-terminal part of M1 protein molecule (residues 2-158) but not for its C-terminal domain (159-252). In the present work M1 protein of A/Puerto Rico/8/34 (H1N1) virus strain in acidic solution was investigated with the help of tritium bombardment. Tritium label incorporation into M1 protein domains preferentially labeled the C-domain and inter-domain loops. Analytical centrifugation and dynamic light scattering experiments demonstrated increased hydrodynamic parameters (diameter) that may be explained by low degree of M1 structural organization. Computational analysis of M1 protein by intrinsic disorder predictions methods also demonstrated the presence of unfolded regions mostly in the C-domain and inter-domain loops. It is suggested, that influenza virus M1 polyfunctionality in infected cell is determined by its tertiary structure plasticity which in its turn results from the presence of unstructured regions.