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At least 361 records · Page 20Linked to original sources
PubMed2003

Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates.

Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.

Adenoviridae↗
PubMed2002

[Z-form of intraphage DNA].

The bacteriophage lambda gt10 DNA containing an insertion of 20 pairs of GC-bases capable of being arranged as Z-form was cloned. Two independent methodological approaches based on the main properties of Z-form were used to study the shape of the insertion: formation of transition bridges composed of unpaired nucleotides between left-rotating Z-forms and right-rotating B-forms of helix (j-domain) and high immunogenic activity of Z-form. O-beta-diethylaminoethylhydroxyamine (OHA), an analogue of hydroxylamine, is capable of reacting specifically with unpaired cytidines. In this work this modification was used to inhibit the process of restriction at BamH1-site adjacent to (gc)10 insertion, that N-Methyl-bis(2-chloethyl) amine (MBCA) is capable of fixing the Z-form of the insertion in situ. Fixed Z-form is conserved even after DNA has been isolated from bacteriophage, thereby providing an opportunity of its identification by anti-Z-antibodies. It was shown that from 4 to 6% of the total number of insertions are in the Z-form. The hypothesis of significant role of Z-form in the process of packing of DNA molecules in capsid is put forward.

Bacteriophage lambda↗
PubMed1990

Constraints in simian virus 40 (SV40) encapsidation, as determined by SV40-based shuttle viruses.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

Animals↗
PubMed1996

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Adenovirus E1 Proteins↗
PubMed2005

Essential amino acids of the hantaan virus N protein in its interaction with RNA.

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

Amino Acid Sequence↗
PubMed1985

Nuclear matrix modifications at different stages of infection by herpes simplex virus type 1.

In BHK-21 cells infected with herpes simplex virus type 1 many virus-induced proteins were found attached to the nuclear matrix. To understand the role of this cell fraction during virogenesis, matrix-associated proteins were analysed at different stages of infection. All the immediate-early protein species were bound to the nuclear matrix and their association with this structure was stable. During the first few hours of infection, the pattern of virus-induced proteins attached to the nuclear matrix remained identical, indicating that polypeptides from the early group are not associated with this cell fraction. Among the late proteins, which are generally structural proteins, 60% of the nuclear proteins were tightly bound to the nuclear matrix. This suggests that the nuclear matrix is involved in at least two different events during virogenesis, regulation of viral infection and assembly of viral capsids.

Animals↗
PubMed1975

Dengue virus-induced modifications of host cell membranes.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.

Cell Line↗
PubMed1990

Adapting the polymerase chain reaction to a double-stranded RNA genome.

We have adapted the polymerase chain reaction (PCR) to a double-stranded RNA (dsRNA) target without possessing unambiguous sequence information. Infectious bursal disease virus of chickens, a member of the binavirus group, has a dsRNA genome which is resistant to denaturation and subsequent enzyme modification. The only published sequence information was for a strain of virus unavailable to us. We have used a quick primer binding assay to select appropriate primers and have combined a simple denaturation method with reverse transcription and subsequent polymerization using the cDNA template to yield amplified product easily detectable by ethidium bromide staining. By varying the times of denaturation, annealing, and polymerization and by reducing the total number of amplification cycles, artifacts have been eliminated when using purified genome as the template. This allowed us to obtain partial sequence information for one viral strain. We have enhanced the utility of our method by optimizing a rapid cell lysis and capsid digestion protocol such that no purification steps are required from initial tissue handling through final PCR product. Total time for all procedures involved no more than 6 h. This technique should be applicable to all other members of the Birnaviradae family and to any other species of dsRNA.

Animals↗
PubMed1994

Flock house virus: a simple model for studying persistent infection in cultured Drosophila cells.

Flock house virus (FHV), isolated from twenty Drosophila melanogaster cell lines, persistently infected with the virus, were examined during successive serial passages by plaque assay and sequence analysis. No phenotypic or genotypic changes in the virus were observed during the establishment of persistent infection, suggesting that it was a cellular modification that led to the first step in establishing the persistent state. Once this state was initiated, the virus was relieved of the need for a functional coat protein to propagate itself and mutations began to accumulate selectively in RNA2, the gene for the coat protein. These changes were manifested by a gradual drift to a smaller plaque population. The replicase activity, coded by RNA1, remained unaltered.

Animals↗
PubMed1999

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus.

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.

Capsid↗
PubMed2006

Interactions of HIV-1 Gag with assembly cofactors.

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.

Amino Acid Sequence↗
PubMed1996

Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding.

A theoretical model is presented that accounts for the facilitation of the pressure dissociation of R17 phage, and for the partial restoration of the concentration dependence of the dissociation, by the presence of subdenaturing concentrations of urea. As an indifferent osmolyte urea should promote the stability of the protein aggregates under pressure, and the decrease in pressure stability with urea concentration demonstrates that such indirect solvent effects are not significant for this case, and that the progressive destabilization is the result of direct protein-urea interactions. By acting as a "homogenizer" of the properties of the phage particles, urea addition converts the pressure-induced deterministic dissociation of the phage into a limited stochastic equilibrium. The model establishes the origin of the uniform progression from the stochastic equilibrium of dimers, to the temperature-dependent and partially concentration-dependent association of tetramers, to the fully deterministic equilibrium observed in many multimers and in the virus capsids.

Bacteriophages↗
PubMed2003

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery.

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology. Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.

Animals↗
PubMedDate not supplied

[2 forms of influenza virus nucleocapsid protein (NP) in virions and infected cells].

Two forms of the major nucleocapsid protein (NP) of influenza virus were found which an polyacrylamide gel electrophoresis had the mobility corresponding to those of proteins with moleculr weights of 56,000 (NP56) and 53,000 (NP53) daltons. A pulse-chase experiment showed the NP53 polypeptide to be a transformation product of NP56 polypeptide. Peptide mapping indicated that both proteins were quite similar, except that NP56 had two additional peptides suggesting the mechanism of NP53 derivation from NP56 by cleavage of a small peptide (molecular weight about 3000 daltons). Both classes of NP protein molecules were found both in infected cells and in virions. The intensity of intracellular modification NP56 leads to NP53 was dissimilar in different cell lines and practically did not depend on the virus strain. In virus preparations obtained from different cells in NP53 protein content varied but this polypeptide was always found as a minor component. In the infected cells the ratio of NP53 to NP56 polypeptides was significantly higher than in virions indicating the preferable incorporation into the virus progeny of polypeptides with intact NP56 protein.

Animals↗
PubMed1985

Site-specific phosphorylation of avian retrovirus nucleocapsid protein pp12 regulates binding to viral RNA. Evidence for different protein conformations.

Phosphorylation of serine 40 of the major nucleocapsid protein of avian retroviruses, pp12, regulates binding to viral RNA (Leis, J., Johnson, S., Collins, L. S., and Traugh, J. A. (1984) J. Biol. Chem. 259, 7726-7732). The phosphorylation state of the protein can be altered in vitro, resulting in the interconversion of the protein between a state of high affinity for single-stranded RNA and low affinity for single- or double-stranded RNA. The reversible phosphorylation of serine 40 is accompanied by a change in the conformation of the protein as demonstrated by quenching of intrinsic tryptophan fluorescence and chemical modification studies. Quenching of fluorescence of the sole tryptophan residue, Trp 80, by poly(U), KI, and CsCl indicates that the microenvironment of this residue is more positive in pp12 than in p12. Chemical modification studies indicate that the 3 lysine residues at positions 36, 37, and 39 of pp12 react with 2,4,6-trinitrobenzenesulfonic acid, while only 1 of these residues reacts in p12. The addition of single-stranded, but not double-stranded RNA, to pp12 protects 2 of the 3 lysine residues from chemical modification, suggesting that the two protected lysyl groups are required for binding to single-stranded viral RNA. In contrast to the phosphorylation of serine 40, phosphorylation of serine 43, catalyzed by protease-activated kinase II in vitro, does not induce changes in the protein conformation nor does it alter the RNA binding properties of the protein.

Amino Acid Sequence↗
PubMed1992

[A new modification of the horse erythrocyte agglutination test for the detection of heterophilic antibodies in infectious mononucleosis].

The new modification (HEA test) is based on the presence of a singularly distinct agglutination of native horse erythrocytes in a serum dilution 1:10 and more from cases with Epstein-Barr virus infectious mononucleosis. In 1665 sera the HEA test was equally often positive as the OCH test (in 5.2%) whereas the IM test with formal-treated horse erythrocytes was positive only in 3.7%. The sensitivity of the IM test in comparison with the OCH test was 61%, the specificity 99.5%. The sensitivity of the HEA test in comparison with the OCH test was 76%, the specificity 98.7%. The specificity of the HEA test in a group of 52 sera without IgG antibodies to EB-viral capsid antigen (EB VCA) was 94%, in another group of 130 sera from hepatitis cases it was 98%. In 103 pairs of sera from infants aged 1-5 years the positivity of the OCH test, HEA test, IgM antibodies to EB VCA and the four-fold rise in the titre of EB-viral IgG were, 20%, 16%, 23% and 24% respectively. Analogous values in 159 pairs of sera from older subjects were 42%, 40%, 38% and 29%. The HEA test is recommended as useful adjunct to the OCH test for the detection of heterophile antibodies even in young children.

Agglutination Tests↗
PubMedDate not supplied

Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.

Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.

Bacterial Proteins↗
PubMed2001

Structural analysis of the -1 ribosomal frameshift elements in giardiavirus mRNA.

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that required a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implicated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a -1 ribosomal frameshift in the GLV transcript.

Giardiavirus↗