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At least 613 records · Page 34Linked to original sources
PubMed1996

The fragmentation of incoming Semliki Forest virus nucleocapsids in mosquito (Aedes albopictus) cells might be coupled to virion uncoating.

The fate of Semliki Forest virus (SFV) nucleocapsid, especially the capsid protein (C-protein), was investigated during the early stages of a productive infection in mosquito Aedes albopictus cells. Infection of the cells resulted in a time dependent accumulation of a C-protein derived fragment. This fragmentation of incoming viral nucleocapsid was prevented by NH4Cl, an agent generally used to elevate the pH in acidic intracellular compartments, suggesting that a low intravesicular pH is required for this process. Density gradient analysis of the postnuclear cell lysate demonstrated that the fragmentation was associated with a cellular compartment showing a density of 1.14 +/- 0.02 g/ml. This cellular compartment was devoid from a lysosomal marker enzyme and represented the timely preceding cellular fraction through which SFV passed before encountering a lysosomal fraction. Furthermore, the intracellular distribution of the viral, 3H-uridine-labeled RNA suggested that the same fraction might represent a key cellular compartment in which the separation of the viral RNA from the viral structural proteins is primed. In conclusion, these data lead to the suggestion that the fragmentation of incoming SFV nucleocapsids in Aedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection.

Aedes↗
PubMed1966

Effect of hydroxyurea on virus development. I. Electron microscopic study of the effect on the development of bacteriophage T4.

Margaretten, William (College of Physicians and Surgeons of Columbia University, New York, N.Y.), Councilman Morgan, Herbert S. Rosenkranz, and Harry M. Rose. Effect of hydroxyurea on virus development. I. Electron microscopic study of the effect on the development of bacteriophage T4. J. Bacteriol. 91:823-833. 1966.-Double fixation in gluteraldehyde and osmium tetroxide and the application of lead staining revealed details of viral structure not previously observed in thin sections. Bacteriophage presumed to have injected its deoxyribonucleic acid (DNA) exhibited a dense, hollow, disc-shaped core. Within the cytoplasm of infected bacteria, the peripheral membrane of the viral heads was clearly visible. Aberrant forms containing the hollow core and believed to be devoid of DNA were encountered in studies of the normal course of development. Hydroxyurea, which is believed to interfere with the production of infective bacteriophage by inhibiting DNA synthesis, resulted in the appearance of viral particles with the hollow disc or with bizarre, distorted cores. However, a significant number of viral heads looked entirely normal and presumably contained a full complement of DNA. Hypotheses are presented to explain these observations.

Coliphages↗
PubMed1982

Coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins.

We examined the synthesis of viral structural proteins in cultured cells infected with the avian coronavirus infectious bronchitis virus. Tryptic peptide mapping was used to determine the structural relationships of the intracellular proteins to the virion polypeptides. Pulse-chase experiments were performed to identify precursors to the virus-specific proteins. We found that the nucleocapsid protein, P51, and the small viral membrane proteins GP31, GP28, and P23 do not undergo post-translational proteolytic processing. In contrast, GP90 and GP84, the two large virion membrane proteins, were found to be produced by cleavage of a single precursor, GP155. This demonstrated that at least one coronavirus mRNA specifies two virion proteins.

Animals↗
PubMed1981

Inactivation of poliovirus.

Inactivation of Polioviruses has been studied extensively in the 1950s and 1960's. It was shown that, to a certain extent, the kinetics of inactivation depend on the viral structure with which the inactivating agents react. Polio-RNA will loose its capacity for multiplication after one hit of an appropriate agent, thus being inactivated in a first order reaction. Multiple hits on the viral protein are needed to render the virus non-proliferate, which is characterized by a second order reaction. Examples for both types of reaction are given. While the "classical" inactivating chemicals are still in use for poliovaccines, other RNA viruses have successfully been treated with other agents and experimentally yet different substances have been described. Aiming for the highest possible antigenicity, the least harmful procedure seems essential. Since the inactivation procedure includes factors other than chemicals, such as temperature, time, pH etc., they too are part of the discussion evaluating the benefits and the shortcomings of the various technologies.

Antigens, Viral↗
PubMed1993

Role of nuclear pore complex in simian virus 40 nuclear targeting.

Cytoplasmically injected simian virus 40 (SV40) virions enter the nucleus through nuclear pore complexes (NPCs) and can express large T antigen shortly thereafter (J. Clever, M. Yamada, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 88:7333-7337, 1991). The nuclear import of the protein components of introduced SV40 was reversibly arrested by chilling and energy depletion, corroborating our previous observation that the nuclear entry of injected SV40 is blocked in the presence of wheat germ agglutinin and an antinucleoporin monoclonal antibody (mAb414), general inhibitors of NPC-mediated import. The nuclear accumulation of virion protein components and large T antigen in nonpermissive NIH 3T3 cells was similar to that in the permissive host, indicating that the ability to use NPCs as a route of nuclear entry appears to be a general property of the injected virus. Injected virions were capable of completing their lytic cycle and forming plaques in permissive cells. During the early phase of SV40 infection, the cytoplasmic injection of mAb414 effectively blocked nuclear T-antigen accumulation for up to 8 h of infection but had very little effect after 12 h of infection. The time-dependent interference with nuclear T-antigen accumulation by the antinucleoporin antibody is consistent with the hypothesis that the infecting virions enter the nucleus through NPCs. The interference study also suggests that the early phase of infection consists of at least two steps: a step for virion cell entry and intracytoplasmic trafficking and a step for virion nuclear entry followed by large-T-antigen gene expression and subsequent nuclear localization of the gene product. Virions were visualized as electron-dense particles in ultrathin sections of samples in which transport was permitted or arrested. In the former cells, electron-dense particles were predominantly observed in the nucleus. The virions were distributed randomly and nonuniformly in the nucleoplasm but were not observed in heterochromatin or in nucleoli. In the latter cells, the electron-dense particles were seen intersecting the nuclear envelope, near the inner nuclear membrane, and in NPCs. In tangential cross sections of NPCs, which appeared as donut-shaped structures, a spherical electron-dense particle was observed in the center of the structure. Immunoelectron microscopy revealed that NPCs were selectively decorated with 5-nm colloidal gold particles-anti-Vp1 immunoglobulin G at the cytoplasmic entrance to and in NPCs, confirming that the morphologically observed electron-dense particles in NPCs contain the viral structural protein. These results support the hypothesis that the nuclear import of SV40 is catalyzed through NPCs by an active transport mechanism that is similar to that of other karyophiles.

3T3 Cells↗
PubMed1990

Comparative studies of hepatitis B virus precore and core particles.

Hepatitis B virus core antigen gene expresses two cocarboxy-terminal proteins, termed precore and core proteins. Both precore and core proteins can form nucleocapsid-like particles. In order to understand the mechanism that leads to the formation of the nucleocapsid, we have expressed precore and core protein sequences in COS cells, a monkey kidney cell line, and compared the properties of these two particles. Our results show that core protein can form particles with various densities and they are present mostly in the cytosol. Precore protein, on the other hand, forms particles with one predominant density, and a majority of these particles are present in the lumen of the endoplasmic reticulum (ER). Furthermore, our results show that, when coexpressed in the same cells, core protein and the ER-associated surface antigens (envelope protein) show colocalization, indicating interaction between these two viral structural proteins.

Animals↗
PubMed1976

Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10.

Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.

Antigen-Antibody Reactions↗
PubMed1986

Pleomorphism of fine structure of rabies virus in human and experimental brain.

Identification of the Negri bodies in the brain of an 8-year-old boy who died 8 days after a paralytic illness and 20 days after a dog bite, and who had received 9 injections of Semple's anti-rabies vaccine, provided evidence that he died of acute rabies encephalitis and not of post-vaccinal allergic encephalomyelitis. The Negri bodies in the human subject and those seen in the inoculated mouse differed in their morphological structure: the former consisted of a matrix of very fine granular material bearing larger granules or strands of higher electron-density resembling nucleic acids and representing products of host cell-virus interaction; and the latter showed better defined areas of granular matrix containing tubular, bullet-shaped and elongated forms of viral structures, and nucleocapsids or capsule-deficient cores, representing the virions, emerging from them. Fine structural examination of the patient's brain and of the inoculated mouse has provided evidence of the pleomorphism of the Negri bodies and the various stages of formation of viral material and virions in them, the animal alone showing the mature virions of rabies, and proving the infectivity of the Negri bodies of the human brain.

Animals↗
PubMed2004

Does Toll-like receptor 3 play a biological role in virus infections?

The Toll-like receptor (TLR) family functions to recognize conserved microbial and viral structures with the purpose of activating signal pathways to instigate immune responses against infections by these organisms. For example, in vitro studies reveal that the TLR3 ligand is a double-stranded RNA (dsRNA), a product of viral infections. From this observation, it has been proposed that TLR3 is likely an important first signal for virus infections. We approached this issue by investigating the role of TLR3 in four different infectious viral models (lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), murine cytomegalovirus (MCMV), and reovirus) and in TLR3 genetically deficient ((-/-)) mice. Our results indicate that TLR3 is not universally required for the generation of effective antiviral responses because the absence of TLR3 does not alter either viral pathogenesis or impair host's generation of adaptive antiviral responses to these viruses.

Animals↗
PubMed1984

Translation in vitro of hepatitis A virus RNA.

RNA extracted from highly purified hepatitis A virus (HAV) particles was translated in an RNA-dependent, cell-free reticulocyte lysate system, and the products were analyzed by SDS-polyacrylamide gel electrophoresis and compared with proteins translated from RNA of poliovirus. The efficiency of translation in vitro of HAV RNA was comparable to that of poliovirus RNA. Processing of the putative precursor molecules of HAV proteins, studied in pulse-chase experiments, was impaired when compared with that of poliovirus. By radioimmunoprecipitation it was shown that most proteins produced by translation in vitro were related immunologically to the viral structural proteins of the virion.

Animals↗
PubMed1993

A gene encoding a highly expressed spindle body protein of Heliothis armigera entomopoxvirus.

The gene encoding the most abundant protein of purified preparations of Heliothis armigera entomopoxvirus (HaEPV) has been cloned and sequenced. The gene sequence encodes a 40.1K polypeptide with a putative N-terminal 20 amino acid leader peptide, and a single potential N-glycosylation site. Analysis of the protein, which has an apparent M(r) of 50K on polyacrylamide gels, confirmed post-translational loss of the leader peptide, but showed no evidence of glycosylation. The protein is related to others previously described from Choristoneura biennis EPV (63% identity) and Autographa californica nuclear polyhedrosis virus (42% identity). Polyclonal antiserum raised against a bacterial fusion protein containing the majority of the HaEPV protein specifically labelled HaEPV spindle bodies; confocal laser scanning microscopy suggests that the protein is distributed throughout those viral structures.

Amino Acid Sequence↗
PubMed2000

Establishment of a rescue system for canine distemper virus.

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

Animals↗
PubMed1990

The T open reading frame of human immunodeficiency virus type 1.

Sequence analysis of multiple isolates of the human immunodeficiency virus type 1 (HIV-1) reveals the existence of a conserved open reading frame, designated T, that partially overlaps the tat, rev, and vpu coding sequences. Here we show that in vitro translation of RNA derived from this region of the viral genome yields a 17 kDa fusion protein, the result of a minus one frameshift event in the overlap between the tat and T open reading frames. It is also shown that messenger RNA species accumulate in HIV-1 infection from which the 17 kDa protein can be made. These observations suggest that ribosomal frameshift events may result in the biosynthesis of viral regulatory as well as viral structural proteins.

Amino Acid Sequence↗
PubMed1989

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.

DNA Mutational Analysis↗
PubMed2005

Glycosylation of beet western yellows virus proteins is implicated in the aphid transmission of the virus.

Beet western yellows virus relies on the aphid M. persicae for its transmission in a persistent and circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary gland epithelial barriers by a transcytosis mechanism where vector receptors interact with virions. The aphid and the peptidic viral determinants implicated in this interaction mechanism have been studied. In this paper, we report that the coat and the readthrough proteins that constitute the capsid of this virus are glycosylated. Modification of the glucidic core of these structural viral proteins by oxidation with sodium metaperiodate or deglycosylation with N-glycosidase F or alpha-D-galactosidase abrogates the aphid transmission of the virus. Aphid transmission could also be inhibited by lectins directed against alpha-D-galactose when aphids were allowed to acquire virus on artificial membranes. These results suggest that the glucidic cores of the capsid proteins of beet western yellows virus contain alpha-D-galactose residues that are implicated in virus-aphid interaction and promote aphid transmission of the virus.

Amino Acid Sequence↗
PubMed2004

RNA sequences in the Moloney murine leukemia virus genome bound by the Gag precursor protein in the yeast three-hybrid system.

Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, Psi. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The Saccharomyces cerevisiae three-hybrid assay was used to directly examine the interaction between MMLV Gag and Psi. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3' to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within Psi supported strong Gag interaction. Instead, an RNA comprised of nearly the entire Psi region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.

Amino Acid Sequence↗
PubMed1997

Probing the structure of the HIV-1 Rev trans-activator protein by functional analysis.

Human immunodeficiency virus type 1 (HIV-1) encodes a trans-acting regulatory protein, termed Rev, which is critically required for virus replication. Rev is a sequence-specific RNA binding protein which mediates the nuclear export of unspliced and incompletely spliced viral mRNAs encoding the viral structural proteins. While CD and fluorescence measurements have provided several possible structural models of Rev, all attempts employing X-ray crystallography and NMR techniques have so far failed to provide more accurate data. We present a new approach to validate alternative structural models of the N-terminal region of Rev which contains the nuclear localization/RNA binding domain. Points of contact between structural elements in a protein were determined by introduction of targeted amino acid substitutions and subsequent scoring of the biological activities. Our data resulted in the suggestion of a new and more refined model of HIV-1 Rev structure which to date has been impossible to obtain by other means.

Animals↗