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At least 1,045 records · Page 58Linked to original sources
PubMed2001

Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency.

Cationized human serum albumin (cHSA) could serve as a potential non-viral vector system for gene delivery. Native human serum albumin was cationized by covalent coupling of hexamethylenediamine to the carboxyl groups resulting in a shift of the isoelectric point from pH 4-5 to 7-9. The cationized albumin underwent spontaneous self-assembly with DNA as demonstrated by retardation of CMV-nlacZ plasmid in agarose gel electrophoresis. Photon correlation spectroscopy showed a decrease of complex size with increasing cHSA/plasmid ratios. Under optimized conditions complexes were formed with 230-260 nm mean diameter and a homogenous, narrow size distribution. At room temperature complexes were stable in 0.9% sodium chloride solution pH 7.4 for 1 h without aggregation. Process parameters such as albumin concentration, incubation time, temperature, pH, order of reagent addition, the presence of bivalent ions and the ionic strength of the complexation medium all influenced the complex size. Confocal laser scanning microscopy showed interactions of a Texas Red labeled cationized albumin with cell membranes of ECV 304 cells and an enhanced endocytic uptake compared to native albumin. The potential for introducing exogeneous DNA into cells was shown using NIH 3T3 fibroblasts. Successful, albeit low reporter gene expression could be achieved in the presence of chloroquine. Under in vitro conditions no toxic effect could be observed. In conclusion, cationized albumin may have promise as a non-toxic vector for gene delivery, especially for DNA vaccination.

DNA↗
PubMed2005

Regulation of translation by ribosome shunting through phosphotyrosine-dependent coupling of adenovirus protein 100k to viral mRNAs.

Adenovirus simultaneously inhibits cap-dependent host cell mRNA translation while promoting the translation of its late viral mRNAs during infection. Studies previously demonstrated that tyrosine kinase activity plays a central role in the control of late adenovirus protein synthesis. The tyrosine kinase inhibitor genistein decreases late viral mRNA translation and prevents viral inhibition of cellular protein synthesis. Adenovirus protein 100k blocks cellular mRNA translation by disrupting the cap-initiation complex and promotes viral mRNA translation through an alternate mechanism known as ribosome shunting. 100k protein interaction with initiation factor eIF4G and the viral 5' noncoding region on viral late mRNAs, known as the tripartite leader, are both essential for ribosome shunting. We show that adenovirus protein 100k promotes ribosome shunting in a tyrosine phosphorylation-dependent manner. The primary sites of phosphorylated tyrosine on protein 100k were mapped and mutated, and two key sites are shown to be essential for protein 100k to promote ribosome shunting. Mutation of the two tyrosine phosphorylation sites in 100k protein does not impair interaction with initiation factor 4G, but it severely reduces association of 100k with tripartite leader mRNAs. 100k protein therefore promotes ribosome shunting and selective translation of viral mRNAs by binding specifically to the adenovirus tripartite leader in a phosphotyrosine-dependent manner.

Adenoviridae↗
PubMed1987

[Development of an expert system as a teaching aid in the serological diagnosis of acute viral hepatitis].

The serological diagnosis of acute viral hepatitis is complex for both practitioners and students. We have developed an expert system to teach the serological diagnosis of acute viral hepatitis in order to help non-specialists in deciding on the diagnosis. Three levels of increasing difficulty have been devised. The procedure consists of the following steps: choice of first-line examinations; discontinuation of the examinations in cases where the diagnosis is ascertained; choice of the most discriminant examination in cases where the diagnosis is doubtful. The value of this computer-assisted teaching must be confirmed by randomized studies involving a group of students trained by "classical" methods and a group of students trained by a computer-assisted method.

Diagnosis, Computer-Assisted↗
PubMed2003

Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection.

To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre-replication complex. Antibody to Ki-67, a conventional marker of cell division, was also studied. Eighty-seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV-infected patients and the proportion of hepatocytes expressing Mcm-2 (3-40%) always exceeded that expressing Ki-67 (1-14%) and correlated positively with increasing stage of fibrosis (P = 0.0001) and viral replication (P = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm-2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm-2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol (P = 0.0067). The proportion of Ki-67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm-2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm-2 is a more sensitive marker of hepatocyte proliferation than Ki-67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.

Adult↗
PubMed2004

Subviral DNAs associated with geminivirus disease complexes.

Ageratum conyzoides (ageratum) is a common and widespread weed species that may act as a reservoir host for geminivirus diseases. Ageratum plants growing in Singapore and exhibiting yellow vein disease are infected with a complex mixture of viral, subviral and recombinant DNA components. This whitefly-transmitted disease is caused by the monopartite begomovirus ageratum yellow vein virus (AYVV) in association with a recently discovered satellite component referred to as DNA beta. Diseased plants also contain a subviral component, referred to as DNA 1, that has probably become associated with the begomovirus and adapted to whitefly transmission during mixed infection with an aphid-transmitted nanovirus. Unlike DNA beta, the nanovirus-like component is not essential for the disease. Recombination between the viral and subviral DNAs occurs frequently and may play an important evolutionary role by generating component diversity. The identification of a similar complex associated with cotton leaf curl disease (CLCuD), a serious constraint to cotton growing in Pakistan, and the isolation of DNA beta homologues from diverse plant species growing in widespread geographical locations suggests that such disease complexes are common and may have a significant impact on agriculture in the eastern hemisphere.

Ageratum↗
PubMedDate not supplied

The H-2 complex regulates both the susceptibility to mouse viral lymphomagenesis and the phenotype of the virus-induced lymphomas.

Neonatal infection of C57BL/10 mice with cloned ecotropic and/or dualtropic mink cell focus-inducing (MCF) mouse leukaemia viruses (MuLV), induces a wide spectrum of different lymphomas of T, B, and non-T/non-B cell types. The H-2 complex has a marked influence on both the development of lymphoma incidence and lymphoma type. A study using the oncogenic MCF 1233 virus and a series of B10 congenic mice enabled the mapping of the following: Resistance to the early development of T cell lymphoma is controlled by the H-2I-A locus. Susceptibility to early T cell lymphomagenesis is associated with an I-A-regulated low anti-MCF 1233 envelope antibody response and persistent infection of the thymus. B10 (H-2b) mice, which are resistant to early T cell lymphomagenesis induced by MCF 1233 or other MuLV isolates, have high anti-MuLV envelope antibody responses which are I-A-regulated. These mice develop more B cell lymphomas late in life in contrast to the early development of T cell lymphoma in B10.A (H-2a) mice. The possible response mechanisms which underlie these observations, including: I-A-regulated immunoselection against MuLV antigens expressed by (pre) leukaemic T cells, aberrant expression of class II MHC antigens on some B cell lymphomas and I-A-regulated chronic immunostimulation of MuLV-expressing (pre) leukaemic B cells, are discussed.

Animals↗
PubMed2002

tRNA(Lys3): the primer tRNA for reverse transcription in HIV-1.

During the assembly of HIV-1, tRNA(Lys) isoacceptors are selectively packaged into the virion, and one of these, tRNA(Lys3), is annealed to the viral RNA genome where it acts to prime the reverse transcriptase (RT)-catalyzed synthesis of viral DNA. We review herein what is known about the selective packaging and annealing of primer tRNA(Lys3). Current evidence suggests that a complex of two major precursor viral proteins, Pr55gag and Pr160(gag-pol), interact with a tRNA(Lys)/lysyl-tRNA synthetase (LysRS) complex during viral assembly, with Pr55gag interacting with both LysRS and Pr160(gag-pol), and RT sequences within Pr160(gag-pol) binding to tRNA(Lys). LysRS appears to target the tRNA(Lys) isoacceptors for incorporation into HIV-1. In the virion, the 3' terminal 18 nucleotides of tRNA(Lys3) anneals to an 18-nucleotide sequence at the 5' terminal region of viral RNA termed the primer binding site (PBS). Evidence is presented that other regions on the tRNA(Lys3) also anneal with other regions in viral RNA upstream of the PBS, resulting in a destabilized tRNA(Lys3) structure. Both viral and tRNA(Lys3) regions need to be denatured to establish annealing, and the roles of both viral and cellular proteins in this process are discussed.

Base Sequence↗
PubMed1998

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.

Biological Transport↗
PubMed1994

Multiple functions of capsid protein phosphorylation in duck hepatitis B virus replication.

We have investigated the role of phosphorylation of the capsid protein of the avian hepadnavirus duck hepatitis B virus in viral replication. We found previously that three serines and one threonine in the C-terminal 24 amino acids of the capsid protein serve as phosphorylation sites and that the pattern of phosphorylation at these sites in intracellular viral capsids is complex. In this study, we present evidence that the phosphorylation state of three of these residues affects distinct steps in viral replication. By substituting these residues with alanine in order to mimic serine, or with aspartic acid in order to mimic phosphoserine, and assaying the effects of these substitutions on various steps in virus replication, we were able to make the following inferences. (i) The presence of phosphoserines at residues 245 and 259 stimulates DNA synthesis within viral nucleocapsids. (ii) The absence of phosphoserine at residue 257 and at residues 257 and 259 stimulates covalently closed circular DNA synthesis and virus production, respectively. (iii) The presence of phosphoserine at position 259 is required for initiation of infection. The results implied that both phosphorylated and nonphosphorylated capsid proteins were necessary for a nucleocapsid particle to carry out all its functions in virus replication, explaining why differential phosphorylation of the capsid protein occurs in hepadnaviruses. Whether these differentially phosphorylated proteins coexist on the same nucleocapsid, or whether the nucleocapsid acquires sequential functions through selective phosphorylation and dephosphorylation, is discussed.

Alanine↗
PubMed2000

Molecular analysis of cerebrospinal fluid: potential for the study of HIV-1 infection of the central nervous system.

The molecular analysis of cerebrospinal fluid (CSF) provides an inestimable tool for the study of HIV infection of the central nervous system (CNS). Current nucleic acid amplification techniques enable the measurement of CSF HIV-1 RNA levels which can be predictive of HIV-associated neurological damage. CSF HIV-1 RNA levels do not necessarily correlate with the corresponding plasma levels, thus supporting the possibility of an intrathecal virus production, i.e., from brain macrophages. However, in early stages of HIV infection, as well as during some opportunistic CNS diseases, CNS or CSF infiltrating lymphocytes might be the main source of CSF virus. A drastic decrease in CSF viral load is usually observed along with a decrease in plasma levels in patients receiving highly active antiretroviral therapy (HAART), with durable suppression of CSF viral load over months. However, during the first weeks of therapy, the dynamics of response may differ in the CSF as compared to plasma, again suggesting that virus replication may be compartmentalised in the CSF. A number of mechanisms are likely to be involved in the response to therapy in CSF, including among the others the trafficking of cell populations supporting viral replication between blood, CNS and CSF, and the role of the anatomical brain barriers in limiting the access of antiretroviral drugs into the CSF. A potential risk associated with compartmentalisation of HIV infection is of an incomplete suppression of virus replication in the CSF, thus creating the ground for local development of anti-HIV drug resistance. In order to assess this occurrence, long-term studies of viral load and genotypic analyses on paired CSF and plasma will be necessary and these will also help elucidate the complex interrelationship between viral replication in these compartments.

Anti-HIV Agents↗
PubMed1996

"HIV-peplotion vaccine"--a novel approach to protection against AIDS by transepithelial transport of viral peptides to Langerhans cells for long-term antiviral CTL response. (A review).

Viral vaccines which stimulate the humoral immune response in humans have been successful in preventing most of the known virus diseases except dengue fever, respiratory syncytial virus infections and HIV-1-related AIDS. Burke [1] raised a concern that anti-HIV-1 antibodies may add a risk factor to immunized individuals infected with HIV-1. An approach to develop HIV-1 vaccines capable of stimulating anti-HIV-1 cytotoxic T cells requires an understanding of the importance of epidermal and epithelial Langerhans cells (LC). These cells are professional antigen-presenting cells which express HLA class I and class II molecules. Epithelial LC are present in a specific layer in the skin, genitalia and gut and may be accessible to viral antigens by local application in a vehicle for transepithelial transport of viral proteins/peptides (designated "HIV-1 Peplotion vaccine"). This approach is supported by the reports that HIV-1 gp160 in ISCOM induced MHC class I CTL response [2], mixing of cationic lipids with viral proteins formed complexes which were delivered to cell cytoplasm and the degraded peptides stimulated CTLs by HLA class I mechanism [3] and viral proteins encapsulated in pH-sensitive liposomes administered to LC induced primary antiviral CTLs [4]. Current studies in our laboratory deal with (a) selection of the vehicle for transepidermal transport of peptides and the conditions for selective uptake by epidermal LC [5]; (b) computer analysis of HIV-1 proteins to detect the putative proteolytic cleavage peptides with amino acid motifs which allow association with different known HLA class I haplotype molecules on LCs and synthetic peptide uptake from "without" by LC. The "HIV-1 Peplotion vaccine", when developed, will be useful for continual stimulation of antiviral CTLs in uninfected individuals and HIV-1 carriers by repetitive application to skin, genitalia and gut. The "Peplotion vaccine" will be applied by vaccinees, will be affordable for all human-populations and, hopefully, will be highly efficient.

Amino Acid Sequence↗
PubMed1991

Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex.

Cytoplasmic extracts prepared from cells infected with metabolically radiolabeled virions of human immunodeficiency virus type 1 contain viral DNA in association with labeled viral proteins. Viral DNA can be purified from these extracts by gel filtration chromatography and sucrose gradient sedimentation as a part of a nucleoprotein complex containing integrase as the only viral protein detectable by immunoprecipitation and gel electrophoretic analysis. The purified complex contains no detectable gag gene products, including p17, p24, p7, or p6, and contains no additional pol gene products, including the p10 protease, p66 and p51 polymerase, or the p15 RNase H. Nearly all of the purified nucleoprotein complexes are capable of integrating into heterologous DNA targets in vitro. These observations demonstrate that integrase is a component of the human immunodeficiency virus type 1 preintegration complex and suggest that integrase may be the only viral protein necessary for the integration of retroviral DNA.

Attachment Sites, Microbiological↗
PubMed1997

Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase.

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.

DNA, Viral↗
PubMed2000

Purified JC virus T and T' proteins differentially interact with the retinoblastoma family of tumor suppressor proteins.

The amino termini of polyomavirus T antigens contain LXCXE and J domains, which are necessary for binding and inactivating the retinoblastoma family of tumor suppressors. Both of these motifs are found in the JC virus (JCV) early proteins T'(135), T'(136), and T'(165), leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating function of the JCV large T antigen (TAg). To investigate this hypothesis, the three JCV T' proteins were produced in a baculovirus expression system and purified by immunoaffinity chromatography. To facilitate purification, hybridomas that secrete antibodies recognizing amino-terminal epitopes of JCV early proteins were produced. Potential interactions between the early viral proteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAg and T' proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosphorylated species of the cellular proteins and exhibited the highest binding affinity to p107 and the lowest affinity to pRB. TAg and T'(165) bound more pRB and less p107 than did T'(135) and T'(136); T'(165) also bound less p130 than the other three early proteins. Results of these in vitro interactions were compared to those obtained in vivo using POJ cells, a transformed human glial cell line that expresses JCV early proteins, relatively high levels of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylated p130 is detected in the transformed cells, and most of this protein was found in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins.

Animals↗
PubMedDate not supplied

[The modern analysis and prediction of the spread of acute respiratory viral infections].

The weekly dynamics of registered morbidity in the whole complex of acute respiratory viral infections (ARVI) for 1960-1994 was studied. The duration of the annual epidemic cycle of ARVI was taken to be the period from week 26 of the given year to week 25 of the following year. The dynamics of the development of the epidemic process made it possible to distinguish 7 definite stages of the epidemic cycle. The assessment of the state of morbidity at the final stages of the completed epidemic cycle and at the first stages of the following epidemic cycle permits the prediction of the level of ARVI morbidity for the whole following epidemic cycle and the determination of the adequate set of prophylactic measures.

Acute Disease↗
PubMed2001

In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection.

Demyelination in multiple sclerosis and in animal models is associated with infiltrating CD8+ and CD4+ T cells. Although oligodendrocytes and axons are damaged in these diseases, the roles T cells play in the demyelination process are not completely understood. Antigen-specific CD8+ T cell lysis of target cells is dependent on interactions between the T cell receptor and major histocompatibility complex (MHC) class I-peptide complexes on the target cell. In the normal central nervous system, expression of MHC molecules is very low but often increases during inflammation. We set out to precisely define which central nervous system cells express MHC molecules in vivo during infection with a strain of murine hepatitis virus that causes a chronic, inflammatory demyelinating disease. Using double immunofluorescence labeling, we show that during acute infection with murine hepatitis virus, MHC class I is expressed in vivo by oligodendrocytes, neurons, microglia, and endothelia, and MHC class II is expressed only by microglia. These data indicate that oligodendrocytes and neurons have the potential to present antigen to T cells and thus be damaged by direct antigen-specific interactions with CD8+ T lymphocytes.

Animals↗