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At least 1,387 records · Page 77Linked to original sources
PubMed1981

Dual functions of bacteriophage T4D gene 28 product: structural component of the viral tail baseplate central plug and cleavage enzyme for folyl polyglutamates. I. Identification of T4D gene 28 product in the tail plug.

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28(ts) phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28(ts) and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28(ts) and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28(ts) particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28(ts) particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28(ts) revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.

Adsorption↗
PubMed1995

Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.

The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.

Base Sequence↗
PubMed1986

Analysis of pseudopodial structure and assembly with viral projections.

The mechanisms by which cells extend motile pseudopodial projections are still poorly understood. Several fundamental mechanisms have been proposed on the basis of hydrostatic pressure, membrane addition and microfilament reorganization. A common focus of all such mechanisms is the growing tip of a pseudopodium. Yet some basic questions about the nature of the tip in natural pseudopodia remain obscure. However, one class of structure, the virus-tipped projections, often contains a well-defined particle, both morphologically and biochemically, and therefore provides a useful model system for the examination of the tips of cellular projections. In P815 cells the virus-tipped projections are long, thin structures closely resembling filopodia in other cells. The apical virus particle is a retrovirus particle produced by the chronic infection existing in this cell line. In demembranated filopodia, the virus particle retains a tight association with a single actin microfilament. Biochemical analyses indicate that the major retroviral structural polypeptide Pr65 is an actin-binding protein that could provide the anchorage site for the actin filament. The existence of a solid virus particle tethered by an actin filament to the cytoskeleton makes it very unlikely that these projections grow by membrane addition at the tip. The major positive implication is that the apex of a projection does not relinquish its interaction with the submembranous cytoskeleton during growth. Such an arrangement would be compatible with either a hydrostatic-pressure-driven or a cytoskeleton-driven mechanism of filopodial growth.

Actin Cytoskeleton↗
PubMed2004

A single RNA recognition motif in splicing factor ASF/SF2 directs it to nuclear sites of adenovirus transcription.

SR protein ASF/SF2 is a general pre-mRNA splicing factor as well as a regulator of alternative splicing. Data presented here show that ASF/SF2 is efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. At the intermediate stages of a productive infection, ASF/SF2 colocalizes with small nuclear ribonucleoprotein particles (snRNPs), splicing factors in ring-like structures surrounding viral replication centres and, at late stages of the infection, in enlarged speckles. Results presented here demonstrate that ASF/SF2 requires only one of the two RNA-recognition motifs (RRMs) present in the protein for its efficient recruitment to the ring-like structures, where viral pre-mRNAs are transcribed and processed, and that the arginine/serine-rich (RS) domain in ASF/SF2 is both redundant and insufficient for the translocation of the protein to active viral RNA polymerase II genes in adenovirus-infected cells.

Adenoviridae↗
PubMed1976

Purification of a glycoprotein from mouse ascites fluid by immunoaffinity chromatography which is related to the major glycoprotein of murine leukemia viruses. Immunologic and structural comparison with purified viral glycoproteins.

Proteins immunologically related to the major glycoprotein from murine leukemia viruses, gp70, are expressed in the tissue and body fluids of unmanipulated mice in the absence of virus particle production. Cross-reactive gp70 was isolated from ascites fluid of NZB and (NZB X NZW)F1 mice utilizing immunoaffinity chromatography. An enriched antibody preparation bound to Sepharose was used as an immunosorbent to purify gp70 over 600-fold in one step with a 30% recovery. The gp70s from ascites fluid were compared with gp70s purified from laboratory virus strains Friend, Rauscher, Moloney, and Scripps leukemia viruses. Direct antibody binding studies showed that the viral gp70s were more closely related to each other than to the gp70s from ascites fluid. Furthermore, the gp70s from ascites fluid were more reactive with antibody to xenotropic virus than were the gp70s from the laboratory viruses tested. Tryptic peptide map analysis of radioiodinated proteins showed that gp70 from Scripps virus is closely related to gp70 from Moloney virus in primary structure and less closely related structurally to gp70 from Rauscher virus and gp70 from NZB ascites fluid.

Animals↗
PubMed1995

Implications for viral uncoating from the structure of bovine enterovirus.

We have determined the crystal structure of a bovine enterovirus, revealing that the topologies of the major capsid proteins and the overall architecture of the virion are similar to those of related picornaviruses. The external loops joining beta-strands are truncated and the canyon region is partially filled by an extension of the VP3 G-H loop giving the viral capsid a relatively smooth appearance. These changes may have implications for cell attachment. In spite of these differences the virus maintains a hydrophobic pocket within VP1, occupied by a specific 'pocket factor' which appears to be myristic acid. These observations support the proposal that a kinetic equilibrium exists between occupied and unoccupied pocket states, with occupation inhibiting uncoating.

Animals↗
PubMed2001

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.

Amino Acid Sequence↗
PubMed1999

Hepatitis A virus capsid protein VP1 has a heterogeneous C terminus.

Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.

3C Viral Proteases↗
PubMedDate not supplied

[The prevalence of etiological forms of viral hepatitis in Khabarovsk].

For the first time the etiological structure of viral hepatitis in the Far East, in Khabarovsk, has been studied. The leading etiological form of viral hepatitis is hepatitis A. The spread of hepatitis B and nondifferentiated hepatitis is less than that of hepatitis A. The ratio of different etiological forms of virus hepatitis is determined by the epidemiological situation. At the period of epidemic hepatitis A morbidity increases while hepatitis B morbidity decreases. The occurrence of nondifferentiated hepatitis at different periods of observation remains unchanged.

Adolescent↗
PubMed1987

Characterization of genotypic and phenotypic variation in plaque-purified strains of HzSNPV Elkar isolate.

Twenty plaque-purified strains of Heliothis zea S-type nuclear polyhedrosis virus (HzSNPV) were characterized based upon restriction endonuclease digests of viral DNAs, structural protein profiles, and larval melanization response. Each of the 20 strains had a unique genotype. Genomic heterogeneity between strains was localized to 4 regions of the HzSNPV genome. Differences in protein profiles of occluded virus were noted for each strain relative to the wild-type isolate. The strains were separated into 3 groups based upon differences in relative virulence and rate of larval melanization upon death. There was no correlation between differences in pathology and specific alterations in either genotype or structural proteins. The genotype of strain HzS-15 was extensively characterized. Physical maps were constructed for the HzS-15 strain and all of the plaque-purified strains.

Animals↗
PubMed1995

[Relation between structure and anti-viral activity in interferon system].

The interferons (IFN) were discovered in 1957 as biological agents interfering with viral replication. IFNs were initially classified according to their sources as leukocyte, fibroblast and immune IFNs. Both leukocyte and fibroblast IFNs are designated as Type I IFNs and immune IFN as a Type II IFN. There are almost twenty related IFNs which belong to the group of Type I IFN. IFN-alpha 2 and IFN-omega 1 are described as members of the large group of Type I IFN. Both natural IFN-alpha 2 and IFN-omega 1 are isolated from human leukocytes. IFN-alpha 2 carries an O-linked carbohydrate moiety, whereas IFN-omega 1 has an N-linked complex oligosaccharide. Recombinant IFN-alpha 2 expressed in E coli. and Sf-9 cells have been characterized. Moreover, recombinant IFN-omega 1 expressed in CHO cells and Sf-9 cells have also been characterized. In the present study, the recombinant human IFN-alpha 2 and IFN-omega 1 were analyzed for their anti-viral activity compared with native products.

Animals↗
PubMed1992

Pseudo--half-knot formation with RNA.

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.

Base Sequence↗
PubMed1980

Influenza: viral determinants of the pathogenicity and epidemicity of an invariant disease of variable occurrence.

If influenza is a riddle wrapped in mystery inside an enigma, then the viral genes are the riddle, the variable surface antigens for which they code are the mystery, and the course and cause of epidemics the ultimate enigma. Paradoxically, the disease itself has remained a stable and recognizable entity through the years, whether initiated by A/PR/8/34 or A/USSR/90/77 variant viruses. Thus, evolution appears to have preserved the disease but not the virus. Among the questions before us are: (1) Have we become obsessed with differences instead of similarities, and have we overemphasized minor differences in viral (antigenic) structure as epidemic determinants? (2) To what extent do viral antigens reflect selection by population antibody? (3) To what extent is antigenic change the pleiotropic consequence of protein structural alteration for purposes other than escape from specific neutralization? These and other questions are discussed in relating viral form to function.

Animals↗
PubMed1998

Antigen processing of two H2-IEd-restricted epitopes is differentially influenced by the structural changes in a viral glycoprotein.

The factors that influence the intracellular location(s) of MHC class II-restricted epitope loading remain poorly understood. We present evidence that two I-Ed-restricted epitopes of the influenza hemagglutinin (HA) molecule, termed site 1 (S1; encompassing amino acid residues 107-119) and site 3 (S3; encompassing amino acid residues 302-313), are generated in distinct endocytic compartments. By means of an epitope-specific mAb, we show that S1 becomes detectable in late endocytic/lysosomal vesicles; using a mutant cell line, we also show that the presentation of S1 is dependent upon H2-DM expression. In contrast, S3; presentation is H2-DM-independent and appears in early endosomes as a result of acid-induced structural changes in HA. Presentation of both epitopes can be made H2-DM-independent by denaturing HA and made H2-DM-dependent by preventing the acid-induced conformational changes from occurring. These findings indicate that the structural context of a given epitope can determine where it is processed.

Animals↗
PubMed2004

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease.

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Amino Acid Sequence↗
PubMed1996

Structure, interactions and dynamics of PRD1 virus II. Organization of the viral membrane and DNA.

Structure, dynamics and stability of the membrane and double-stranded (ds) DNA genome packaged within the native PRD1 virion have been probed by laser Raman spectroscopy. The Raman signature of PRD1 is complex, but exhibits distinctive marker bands diagnostic of the internal lipid bilayer and dsDNA. The Raman markers demonstrate, respectively, a liquid crystalline lipid phase(L(alpha)) and B DNA conformation throughout the temperature range (5 degrees to 50 degrees) of virion stability. Despite the absence of large scale lipid phase transitions or DNA melting, small temperature-dependent changes in the Raman markers of lipid and DNA are detected, indicating coupling between their structures. Minor deviations of DNA from the canonical B form are imposed by the membrane. The Raman markers indicate further that base stacking and phosphate group interactions of the packaged PRD1 genome differ from those of unpackaged PRD1 DNA. Specific Raman band perturbations are proposed as indicators of DNA-membrane interaction. Hydrogen-deuterium exchange kinetics of packaged and unpackaged PRD1 DNA are indistinguishable, demonstrating that base imino and amino protons are not affected significantly by either the condensation or membrane enclosure associated with DNA packaging. This contrasts with the significant acceleration of base exchanges detected in the packaged DNA of bacteriophage P22, which lacks a viral membrane. The distinctive H-->2H exchange profile of the PRD1 genome, the absence of packaging-induced acceleration of exchange kinetics and the apparent direct interaction between DNA and phospholipids suggest a specific role for the viral membrane in PRD1 assembly. We propose a "membrane-surface-catalyzed" model for dsDNA condensation and organization within the PRD1 virion.

Bacteriophages↗