PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “viral complexity”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources
PubMed1993

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells.

Cytosine↗
PubMed1979

Auto-reaction phenomenon detected by enzyme electrosyneresis in the serum of patients with viral hepatitis and other diseases with immune complexes.

The use of modified electrosyneresis by making 760 sera of healthy persons or persons suffering from various diseases with immune complexes to react with their own pronase-treated serum has shown the following results: - One of 220 sera of healthy persons, 11 were positive in ESE (5%); - Out of 123 sera of HBsAg carriers, 23 were positive (18.6%); - Out of 135 sera of patients with acute viral type B hepatitis, 132 were positive (97.7%); - Out of 168 sera of patients with acute HBsAg negative hepatitis, 127 were positive (75.5%); - 4 cases of fulminant hepatitis were all strongly positive; - 54 cases of patients with rheumatoid arthritis were 100% positive; - 2 cases of patients with systemic lupus erythematosus were positive; - Out of 6 patients with glomerulonephritis 3 were positive; - Out of 34 patients with carcinoma of various organs, 19 were positive (55.88%). The Authors believe, on the basis of the research work set forth partly in the present report, that this interaction is connected with the presence of immune complexes. The method used has been called Enzyme Electrosyneresis (ESE) and the detected antigen "Auto antigen exposed by protease and by electrosyneresis" (AEPE).

Animals↗
PubMed2002

Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription.

Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.

Acid Anhydride Hydrolases↗
PubMed1980

The rolling circle . capsid complex as an intermediate in phi X DNA replication and viral assembly.

Late in the life cycle of the single-stranded DNA phage phi X, the synthesis of positive strand DNA is coupled to the maturation of progeny virions. DNA synthesis and packaging take place in a replication-assembly complex, which we have purified to homogeneity and characterized. The following conclusions can be drawn: 1. The DNA component of the replication-assembly complex is a rolling circle with a single-stranded DNA tail which is less than or equal to genome length. 2. The major protein component of the replication-assembly complex is an intact viral capsid, as shown by gel analysis of 35S-labeled complexes. As replication proceeds at the DNA growing point, the positive strand tail of the rolling circle is displaced directly into the capsid. In addition to the capsid, the complex contains at least 1 molecule of the phi X gene A nicking protein, which appears to be covalently linked to the DNA. 3. The rolling circle . capsid complex can be purified to homogeneity by taking advantage of its uniform sedimentation velocity (35 S) and its uniform density upon equilibrium centrifugation in CsCl (1.50 g/cc). 4. The replication-assembly complex can be visualized in the electron microscope. An electron-dense particle, which has the dimensions of a viral capsid, is observed attached to a rolling circle at the DNA growing point. 5. Hybridization of specific phi X restriction fragments to the deproteinized, single-stranded tails of intact rolling circles has allowed the use of these replicating intermediates to determine both the origin/terminus and the direction of phi X positive strand DNA synthesis. The ends of the rolling circle tails map in the Hae III restriction Fragment Z6b, at the position on the phi X genome at which the gene A endonuclease is known to cut. This result indicates that this endonuclease participates in the "termination" of each round of synthesis by cutting off unit-length viral genomes. 6. Rolling circle . capsid complexes were also isolated from two other icosahedral, single-stranded DNA phages: G4 and St-1. The rolling circle . capsid complex seen in the case of the single-stranded DNA phages represents the first example of a structure in which DNA synthesis and viral assembly occur in a coupled manner. This tight coordination explains why double-stranded DNA circles are the net product of synthesis early in the viral life cycle while only single-stranded DNA circles are produced later. The single-stranded tails of the rolling circle intermediates are available for conversion to the duplex state at early times, whereas the concentration of preformed capsids later is high enough to bind to all of the replicating molecules and package the emerging positive strand DNA.

Bacteriophage phi X 174↗
PubMed2006

Brownian diffusion and surface kinetics of liposome and viral particle uptake by human lung cancer cells in-vitro.

In this study, the simultaneous roles of transport, diffusion, and surface kinetic uptake of liposome (Lip-FD), adenoviral (AD-Cy2), and liposome-adenoviral complex (lip-FD-Ad) particles by a non-small cell human lung cancer (A549) were examined through a coupling of in vitro experimental and mathematical modeling techniques. Experimentally, quantitative fluorescence spectroscopy was used to monitor time dependent particle uptake rates including low temperature (5 degrees C) conditions where endocytosis could be inhibited. Mathematically, analytic solutions to the Brownian particle diffusion equation with Langmuir type boundary conditions for the adsoprtion, desorption, and endocytosis process, were obtained for both unsteady and steady-state (no endocytosis) conditions. By direct comparisons of experimental data to model solutions, the adsorption constants, desorption constants, and number of cell surface receptor sites were determined for all particle types considered. It was found that the particle adsorption and desorption constants were of the same order of magnitude compared to earlier studies (Singh, M., T. Ghose, G. Faulkner, and M. Mezei. Cancer Res. 49:3976-3984, 1990.) using different cell lines, particle types, and methodologies. Also in agreement with previous studies using differing cell lines and methodologies (Miller, C. R., B. Boundurant, S. D. McLeon, K. A. McGovern, and D. F. O'Brien. Biochemistry 37:12875-12883, 1998; Perry, D. G., and W. J. Martin II. J. Immunol. Methods 181:269-285, 1995; Muller, W. J., K. Zen, A. B. Fisher, and H. Shuman. Am. J. Physiol. L11-L19, 1995), the number of cell surface receptor sites was predicted to be several orders of magnitude higher for liposome and liposome-viral complex than for viral particles alone, suggesting a nonspecific or nonrestrictive binding pattern for liposomes and liposome complexes and a specific or restrictive binding pattern for viral particles. The surface kinetic constants obtained here for the A549 cells may be useful in physiological modeling or pharmacokinetic applications of chemical or genetic carrying particles in the treatment of lung cancer and other lung diseases. Furthermore, the methodologies given here are straightforward and can be applied to other particle-cell uptake systems.

Adenoviridae↗
PubMed1969

Membranous structures associated with translation and transcription of poliovirus RNA.

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.

Centrifugation, Density Gradient↗
PubMed1994

Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.

Amino Acid Sequence↗
PubMed1979

Analysis of chromosomes, nucleic acids, and polypeptides in hamster cells transformed by herpes simplex virus type 2.

Syrian hamster embryo fibroblasts were oncogenically transformed by UV-inactivated Herpes simplex type 2. Eighteen clones were isolated shortly after transformation occurred. Two clones and their tumor derivatives were studied using several techniques. The karyotype analysis revealed different chromosome patterns in the two clones and a tendency toward hypodiploidy in the tumor derivatives. All of these cell lines were shown by molecular hybridization to contain 40% of the HSV-genome in several copies. The viral DNA sequence complexity was retained in the tumor derivatives, but a decrease in the copy number was observed. Viral RNA's were detected by in situ hybridization in all the lines that were tested. Viral antigens could be observed in these transformed cells by immunofluorescence. Finally, polypeptide analysis showed three differences between normal and transformed cells.

Animals↗
PubMedDate not supplied

RNA--streptomycin interaction on cellulose.

Bichromatic paper disk spectrophotometry allowed the demonstration of an analogy between the increase in the solubility of highly polymerized RNA complexed with streptomycin cations (which accounts for the preservation in vivo of the infectivity of a viral RNA--streptomycin complex) and the effect of a cellulose/solution interface on the distribution of the highly polymerized RNA.

Cellulose↗
PubMed1995

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Base Sequence↗
PubMed1997

A highly conserved region of the Sendai virus nucleocapsid protein contributes to the NP-NP binding domain.

The nucleocapsid protein (NP) of Sendai virus is an essential component of both the nucleocapsid template and the NP-NP and NP0-P protein complexes required for viral RNA replication. When expressed alone in mammalian cells NP self-assembles into nucleocapsid-like particles which appear to contain cellular RNA. To identify putative NP-NP binding domains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258-357, MBP-NP1) and the N-terminal 255 amino acids (MBP-NP2) of NP both oligomerized, suggesting that these regions contain sequences important for NP-NP self-assembly. In addition, the MBP-NP1 fusion protein can function as an inhibitor of viral RNA replication. Complementary studies involving site-directed mutagenesis of the full-length NP protein have identified specific residues in the CCR which are essential for viral RNA replication in vitro. Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP-NP interaction. A third mutant, NP260-1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, appear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific residues in the CCR of the native NP protein which appear to be important for NP-NP or NP-RNA interactions and for genome replication.

Amino Acid Sequence↗
PubMed2004

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Amino Acid Sequence↗
PubMed1993

Cellular stress response induces selective intranuclear trafficking and accumulation of morbillivirus major core protein.

BACKGROUND: Using canine distemper virus (CDV) encephalomyelitis as a model for morbillivirus-induced intranuclear inclusion body (INB) formation, we previously demonstrated that the cellular basis for INB within infected astrocytes are viral antigen-associated nucleolar derivatives known as complex nuclear bodies. Here, we examined the relationship between the cellular stress response and intranuclear CDV protein trafficking so that we may begin to define cellular events leading to formation of complex nuclear bodies containing viral protein. EXPERIMENTAL DESIGN: Confocal dual-label immunofluorescence microscopy was used to simultaneously determine the distribution of CDV antigen and the major inducible 70 kilodalton (kd) heat shock protein (i.e., 72 kd heat shock protein (HSP)) within infected canine central nervous tissue. This dual-label immunofluorescent technique was also used to test the relationship between cellular stress and CDV core protein distribution in a CDV persistently infected cell line. RESULTS: The cytoplasm of INB-positive astrocytes expressed elevated 72 kd HSP characteristic of the cellular stress response; INB contained 72 kd HSP and the major CDV nucleocapsid protein (i.e., N). Intranuclear inclusion bodies did not stain positively for the CDV nucleocapsid phosphoprotein nor was intranuclear N protein observed in the absence of nuclear or elevated cytoplasmic 72 kd HSP. In the persistently infected cell line, induction of the cellular stress response elevated 72 kd HSP and produced translocation of the CDV N protein to the nucleus from its normal location in the cytoplasm of unstressed infected cells. As observed in vivo, the transport was specific to N and was correlated to the induction of complex nuclear bodies. The latter was documented by transmission electron microscopy, where complex nuclear bodies formed in shocked infected cells, but not shocked uninfected cells or nonshocked infected cells. CONCLUSIONS: CDV infection in vivo induces the cellular stress response. Paradoxically, this normally protective cellular response mediates redistribution of viral N protein from the cytoplasm into infected cell nuclei, which is the likely basis for complex nuclear body formation and a unique form of virus-induced cytopathic effect.

Animals↗
PubMed2001

Dynamics of viral quasispecies during interferon therapy in non responder chronic hepatitis C patients.

BACKGROUND: the reference method to study the HCV complexity was cloning and sequence analysis of a sufficient number of clones. The evolution of the viral complexity in chronic non responder patients during treatment with standard doses of interferon was not very well investigate because this method was expensive and labour intensive when large series of patients were concerned. Meanwhile, with the alternative Single-Strand Conformation Polymorphism (SSCP) method, a rough estimation of the quasispecies present in a given sample could be obtained. OBJECTIVES: the aim of the study was to analyse the evolution of HCV heterogeneity, investigated by SSCP analysis targeted to the HVR-1, in 30 nonresponders chronic hepatitis C patients treated by Interferon-alpha 3MUI. RESULTS: genotype 1 was the main HCV type found in this population (77% of non responder patients). Before treatment, the SSCP assay revealed a high complexity pattern: the median of SSCP band number was 9. During IFN-alpha treatment, SSCP band number didn't change. However a significant decrease of the viral load was observed (P<0.01). Patients with variations in their SSCP patterns after therapy significantly decreased HCV RNA levels (P<0.002). In one third of patients the SSCP profile didn't change at all. CONCLUSIONS: we observed that viral heterogeneity didn't change in non responder chronic hepatitis C patients during IFN-alpha treatment. Nevertheless patients with a low number of pre-treatment quasispecies exhibited an improvement of the response (P<0.02). These phenomena were probably due to a selection of resistant variants present prior onset of therapy.

Adult↗
PubMed2000

Plant DNA viruses and gene silencing.

Gene silencing is a multifaceted phenomenon leading to propagative down-regulation of gene expression. Gene silencing, first observed in plants containing transgenes, can operate both at the transcriptional and post-transcriptional levels. Silencing effects can be triggered by nuclear transgenes and by cytoplasmic RNA viruses, and it can be propagated between these elements and endogenous plant genes that share sequence homology. Although some aspects of gene silencing are becoming better understood, little is yet known about the relationship between nuclear and cytoplasmic events. Plant DNA viruses-- both the ssDNA geminiviruses and the reverse-transcribing pararetroviruses-- have properties with the potential to initiate gene silencing in the nucleus and in the cytoplasm. Characteristics include production of multiple copies of viral DNA genomes in the nucleus, illegitimate integration of viral DNA into host chromosomes mimicking transgene transformation, and generation of abundant viral RNAs in the cytoplasm. Evidence is emerging that geminiviruses and plant pararetroviruses can interact with the gene silencing system either from introduced DNA constructs or during viral pathogenesis. Some observations suggest there are complex relationships between DNA viral activity, transcriptional and post-transcriptional gene silencing mechanisms. DNA viruses also have properties consistent with an ability to counteract the plant silencing response. In this article, features of plant DNA viruses are discussed in relation to gene silencing phenomena, and the prospects for understanding the interaction between nuclear and cytoplasmic silencing processes.

DNA Viruses↗
PubMed2004

Complex signals in the genomic 3' nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA.

The genomes of positive-strand RNA viruses strongly resemble cellular mRNAs. However, besides operating as a messenger to generate the virus-encoded proteins, the viral RNA serves also as a template during replication. A central issue of the viral life cycle, the coordination of protein and RNA synthesis, is yet poorly understood. Examining bovine viral diarrhea virus (BVDV), we report here on the role of the variable 3'V portion of the viral 3' nontranslated region (3'NTR). Genetic studies and structure probing revealed that 3'V represents a complex RNA motif that is composed of synergistically acting sequence and structure elements. Correct formation of the 3'V motif was shown to be an important determinant of the viral RNA replication process. Most interestingly, we found that a proper conformation of 3'V is required for accurate termination of translation at the stop-codon of the viral open reading frame and that efficient termination of translation is essential for efficient replication of the viral RNA. Within the viral 3'NTR, the complex 3'V motif constitutes also the binding site of recently characterized cellular host factors, the so-called NFAR proteins. Considering that the NFAR proteins associate also with the 5'NTR of the BVDV genome, we propose a model where the viral 3'NTR has a bipartite functional organization: The conserved 3' portion (3'C) is part of the nascent replication complex; the variable 5' portion (3'V) is involved in the coordination of the viral translation and replication. Our data suggest the accuracy of translation termination as a sophisticated device determining viral adaptation to the host.

3' Untranslated Regions↗
PubMed1993

Immunological cross-reactions and interactions between the Drosophila melanogaster ref(2)P protein and sigma rhabdovirus proteins.

The ref(2)P gene is one of the Drosophila melanogaster genes involved in the inhibition of sigma rhabdovirus multiplication. The partial restriction of viral replication varies according to the ref(2)P alleles and virus strains and involves intracellular interactions between parasite and host products. We identified the protein encoded by the ref(2)P gene and produced polyclonal antibodies directed against the whole ref(2)P protein obtained from a recombinant baculovirus and against a part of the protein expressed as a fusion protein. These antibodies were used to study the interactions with sigma virus proteins by different immunoprecipitation techniques. We showed that the native ref(2)P protein shared conformation-dependent common epitopes with the viral structural genome-associated N protein. Furthermore, the cellular protein was found to be associated in complexes with the viral P protein required for RNA polymerase activity. The significance of these observations in the control of sigma virus multiplication by its host is discussed.

Animals↗