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At least 991 records · Page 55Linked to original sources
PubMed1987

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation.

We used empty capsids of polyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90-95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.

Capsid↗
PubMed1997

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.

Amino Acid Sequence↗
PubMed2000

The E1 helicase of human papillomavirus type 11 binds to the origin of replication with low sequence specificity.

Expression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types.

Base Sequence↗
PubMed1996

Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs.

Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.

Autoradiography↗
PubMed1985

CSF immune complexes in multiple sclerosis.

Immune complexes from the CSF of 12 individuals (6 with exacerbations of MS and 6 with other neurologic or psychiatric disease) were isolated and characterized. Three MS patients had complexed herpes simplex type I viral antigen and antibody; three patients had complexed myelin basic protein. Two MS patients and one with hypoxic encephalopathy had complexed antibody directed against brain glycolipids. CSF complexes were distinct from serum complexes, suggesting intrathecal origin. Reactivation of latent brain virus may play a role in exacerbations of MS.

Adult↗
PubMed1998

Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered.

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.

Capsid↗
PubMed1991

Herpes and human ribonucleotide reductases. Inhibition by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone (348U87).

The mode of inactivation of herpes simplex virus type 1 and human ribonucleotide reductases by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone++ + (348U87) was determined and compared to that described previously [Porter et al. Biochem Pharmacol 39: 639-646, 1990] for 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U). 348U87 inactivated herpes ribonucleotide reductase faster than did A1110U. Moreover, iron-complexed 348U87 was a considerably more potent inactivator than iron-complexed A1110U. It appeared to efficiently form an initial complex with the viral enzyme prior to rapid enzyme inactivation. The combination of 348U87 and iron-complexed 348U87 inactivated with a rate constant that was slightly greater than the sum of their individual rate constants of inactivation. The corresponding combination of A1110U species inactivated with a rate constant that was much greater than the sum of the individual rate constants of inactivation. Herpes ribonucleotide reductase that had been inactivated by either species of 348U87 was reactivated by diluting the enzyme and inactivators into assay medium containing excess iron. 348U87 was also an effective inactivator of herpes simplex virus type 2 and varicella zoster virus ribonucleotide reductases. The iron-complexed forms of 348U87 and A1110U exhibited very different modes of inactivation of human ribonucleotide reductase. Iron-complexed 348U87 was a tight-binding inactivator, whereas iron-complexed A1110U was only a weak, non-inactivating, inhibitor. Furthermore, the inactivation by iron-complexed 348U87 was not stimulated by either 348U87 or A1110U, whereas the weak inhibition by iron-complexed A1110U was converted to rapid inactivation by A1110U. Excess iron prevented the inactivation by iron-complexed 348U87. Uncomplexed 348U87 was similar to uncomplexed A1110U in that it was not an inhibitor of the human enzyme.

Antiviral Agents↗
PubMed1996

Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm.

All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3' untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.

Animals↗
PubMed1980

Human glomerulonephritis and persistent non-polio enterovirus infection.

In a prospective work, we have studied the non-polio enterovirus (NPEV) excretion in spot urine and stools by four appropriate cell cultures, in four different groups: 20 exposed controls (GI), 88 patients with renal biopsy 'proven GN' (GII), 38 cases with 'proven nonGN' (GIII), and 9 with 'probable nonGN' (GIV). The positive excretion in stools and/or urine is respectively 0 and 5% in Group I, 14 and 33% in Group II, 6 and 19% in Group III, and 13 and 22% in Group IV. Viruria, the consequence of a viraemia, is therefore associated with 'proven GN' (p less than 0.05). In the majority of patients with positive NPEV excretion, we have made an additional but similar study, 3-12 months later. Persistent excretion was confirmed in 22/30 cases in Group II (73%) versus 0/8 in Group III-IV (p less than 0.001). These data concerned patients with membranoproliferative GN [5], membranous GN [6], mesangial IgA GN [6], endocapillary GN [2] or minimal lesions [3]. Thus we have demonstrated a significant relation between persistent NPEV urine/stools excretion and the occurrence of active GN in humans. Such persistent viral infections may represent the cause of some GN, probably mediated by an immune complex mechanism with viral antigens.

Enterovirus↗
PubMed1975

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.

Amino Acids↗
PubMed1996

Viral transactivators E1A and VP16 interact with a large complex that is associated with CTD kinase activity and contains CDK8.

Previously, we showed that the viral transactivator proteins E1A and VP16 specifically interact with a cellular CTD kinase activity in vitro. We now report that E1A and VP16 complexes contain human CDK8, a newly identified member of the cyclin-dependent kinase family that has been shown to be a component of the RNA polymerase II (RNAP II) holoenzyme complex. The presence of CDK8 in the E1A- and VP16-containing complexes is specific for a functional activation domain of these viral transactivators, strongly suggesting that this association is relevant for the transactivation function of E1A and VP16. We show that CDK8 is associated with CTD kinase activity and that CDK8 co-fractionates with E1A- and VP16-associated CTD kinase activity over several chromatography columns. Therefore, CDK8 is likely responsible for the E1A- and VP16-associated CTD kinase activity. Gel filtration chromatography indicates that the E1A- and VP16-associated CTD kinase activity has a molecular size of approximately 1.5 MDa and contains cyclin C and the human homolog of SRB7 in addition to CDK8. This implies that E1A and VP16 associate with the RNAP II holoenyzme. We also looked at the transcriptional activity of CDK8 and found that CDK8 can function as a transcriptional activator when fused to the DNA binding domain of GAL4. Surprisingly, the ability of GAL4-CDK8 to activate transcription in this assay was not dependent on the kinase activity of CDK8, since a kinase-deficient mutant of CDK8 stimulated transcription nearly as well as wild-type GAL4-CDK8. This suggests that CDK8 may play a role in transcription that is distinct from its ability to function as a CTD kinase.

Adenovirus E1A Proteins↗
PubMed1976

Effect of Sarkosyl on chromatin and viral RNA synthesis. The isolation of SV40 transcription complex.

The endogenous RNA polymerase activity of mouse nucleic is enhanced several-fold by the anionic detergent Sarkosyl. The action of Sarkosyl is exerted primarily on the alpha-amanitin sensitive form of the enzyme. This detergent causes the release of nearly all the protein associated with cellular DNA but does not release initiated RNA polymerase. Sarkosyl was also able to activate the RNA polymerase activity from mitotic cells, in which transcription of the highly condensed chromatin is minimal. The use of this anionic detergent has also permitted the extraction of a nucleoprotein complex from Simian Virus 40 (SV40) infected monkey cells. Molecular hybridization experiments have established the viral specificity of the RNA synthesized in vitro by the endogenous polymerase present in this complex.

Animals↗
PubMed1987

Viral gene expression, antibody production and immune complex formation in human immunodeficiency virus infection.

Human immunodeficiency virus (HIV) antigen (HIV-Ag) in polyethylene glycol (PEG) precipitates and supernatants and HIV antibodies (HIV-Ab) to core and envelope antigens were studied in serial serum samples of three HIV-Ab seroconverters and 11 HIV-Ab seropositive men with a mean follow-up time of 16.1 months. In five men not progressing beyond persistent generalized lymphadenopathy (PGL) and two progressing to AIDS, HIV-Ag was detected once in 'free' configuration before HIV-Ab seroconversion and persistently or intermittently 'complexed' thereafter; in six of these men HIV core antibodies were continuously present with a declining level in one. In two men not progressing beyond PGL and five progressing to AIDS HIV-Ag was detected 'complexed' before HIV-Ab seroconversion once and persisted predominantly in 'free' configuration thereafter, while no HIV core antibody was detected in six of these men and a declining level in one. HIV-Ag was detected in 37% (14 out of 38) of HIV core antibody seropositive samples, mostly in 'complexed' form, while HIV-Ag was detected in 86% (43 out of 50) of HIV core antibody seronegative samples, mostly in both 'complexed' and 'free' configuration. Antibodies to HIV envelope antigen were detected in all HIV-Ab seropositive samples. These results indicate that the level of HIV-Ag expression is the primary determinant of detectability of HIV core antigens as well as antibodies. Enhancement of HIV-Ag expression, in a significant number of cases associated with clinical deterioration, appears to lead to clearance of HIV core antibodies in immune complexes, while HIV envelope antibody levels remain relatively unaffected.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis.

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.

Anticodon↗
PubMed2005

Transcellular protein transduction using the Tat protein of HIV-1.

The Tat protein of HIV-1 is a powerful transactivator of gene expression. By interacting with a structured RNA sequence at the 5' end of the viral mRNA, it promotes the remodeling of chromatin and the recruitment of processive RNA polymerase complexes at the viral promoter. In addition to these transcriptional functions, a short amino acid motif, highly enriched in basic amino acids, promotes the export of the protein from the expressing cells. Once in the extracellular environment, the same basic domain of Tat binds to cell surface heparan sulfate proteoglycans; through this interaction, the protein is internalized by a variety of different cell types. Cellular internalization of Tat and Tat fusion proteins requires the integrity of cell membrane lipid rafts and mainly occurs through caveolar endocytosis. The Tat basic domain, when attached to large protein cargos, also mediates their efficient cellular internalization and can be thus utilized for transcellular protein transduction. This property has already been successfully exploited for the delivery of heterologous proteins, nanoparticles, liposomes, phage and viral vectors, and plasmid DNA. The biological significance of intercellular Tat trafficking in the context of viral infection still remains elusive.

Animals↗