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At least 289 records · Page 16Linked to original sources
PubMed2005

Construction of prophylactic human papillomavirus type 16 L1 capsid protein vaccine delivered by live attenuated Shigella flexneri strain sh42.

To express human papillomavirus (HPV) L1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed. HPV type 16 L1 (HPV16L1) gene was inserted into plasmid pHS3199 to form the pHS3199-HPV16L1 construct, and pHS3199-HPV16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV16L1 could be expressed stably in the recombinant strain sh42-HPV16L1. Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles (VLPs) were detected in the sera, intestinal lavage and vaginal lavage from animals immunized by sh42-HPV16L1. The number of antibody-secreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited murine hemagglutination induced by HPV16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig's mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.

Animals↗
PubMed2002

Enhanced response to antibody binding in engineered beta-galactosidase enzymatic sensors.

Peptide display on solvent-exposed surfaces of engineered enzymes allows them to respond to anti-peptide antibodies by detectable changes in their enzymatic activity, offering a new principle for biosensor development. In this work, we show that multiple peptide insertion in the vicinity of the Escherichia coli beta-galactosidase active site dramatically increases the enzyme responsiveness to specific anti-peptide antibodies. The modified enzymes HD7872A and HT7278CA, carrying eight and 12 copies respectively of a foot-and-mouth disease peptide per enzyme molecule, show antibody-mediated activation factors higher than those previously observed in the first generation enzymatic sensors, for HT7278CA being close to 400%. The analysis of the signal transduction process with multiple inserted proteins strongly suggests a new, non-exclusive mechanism of enzymatic regulation in which the target proteins might be stabilised by the bound antibody, extending the enzyme half-life and consequently enhancing the signal-background ratio. In addition, the tested sensors are differently responsive to sera from immune farm animals, depending on the antigenic similarity between the B-cell epitopes in the immunising virus and those in the peptide used as sensing element on the enzyme surface. Altogether, these results point out the utility of these enzymatic biosensors for a simple diagnosis of foot-and-mouth disease in an extremely fast homogeneous assay.

Amino Acid Sequence↗
PubMed2004

Mammalian cell transduction and internalization properties of lambda phages displaying the full-length adenoviral penton base or its central domain.

In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered lambda by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286-393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286-393) includes the principal receptor-binding motif ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda Pb (286-393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.

Bacteriophage lambda↗
PubMed2003

Purification, identification, and biochemical characterization of a host-encoded cysteine protease that cleaves a leishmaniavirus gag-pol polyprotein.

Leishmania RNA virus (LRV) is a double-stranded RNA virus that infects some strains of the protozoan parasite leishmania As with other totiviruses, LRV presumably expresses its polymerase by a ribosomal frameshift, resulting in a capsid-polymerase fusion protein. We have demonstrated previously that an LRV capsid-polymerase polyprotein is specifically cleaved by a Leishmania-encoded cysteine protease. This study reports the purification of this protease through a strategy involving anion-exchange chromatography and affinity chromatography. By using a Sepharose-immobilized lectin, concanavalin A, we isolated a fraction enriched with LRV polyprotein-specific protease activity. Analysis of the active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoreses and silver staining revealed a 50-kDa protein that, upon characterization by high-pressure liquid chromatography electrospray tandem mass spectrometry (electrospray ionization/MS/MS), was identified as a cysteine protease of trypanosomes. A partial amino acid sequence derived from the MS/MS data was compared with a protein database using BLAST software, revealing homology with several cysteine proteases of Leishmania and other trypanosomes. The protease exhibited remarkable temperature stability, while inhibitor studies characterized the protease as a trypsin-like cysteine protease-a novel finding for leishmania. To elucidate substrate preferences, a panel of deletion mutations and single-amino-acid mutations were engineered into a Gag-Pol fusion construct that was subsequently transcribed and translated in vitro and then used in cleavage assays. The data suggest that there are a number of cleavage sites located within a 153-amino-acid region spanning both the carboxy-terminal capsid region and the amino-terminal polymerase domain, with LRV capsid exhibiting the greatest susceptibility to proteolysis.

Animals↗
PubMed2001

Protection of rabbits against rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a potyvirus-based vector.

A new plum pox potyvirus (PPV)-based vector has been constructed for the expression of full-length individual foreign proteins. The foreign sequences are cloned between the NIb replicase and capsid protein (CP) cistrons. The heterologous protein is split from the rest of the potyviral polyprotein by cleavage at the site that originally separated the NIb and CP proteins and at an additional NIa protease recognition site engineered at its amino-terminal end. This vector (PPV-NK) has been used to clone different genes, engendering stable chimeras with practical applications. We have constructed a chimera expressing high levels of jellyfish green fluorescent protein, which can be very useful for the study of PPV molecular biology. The VP60 structural protein of rabbit hemorrhagic disease virus (RHDV) was also successfully expressed by making use of the PPV-NK vector. Inoculation of extracts from VP60-expressing plants induced a remarkable immune response against RHDV in rabbits, its natural host. Moreover, these animals were protected against a lethal challenge with RHDV.

Amino Acid Sequence↗
PubMed2000

Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon.

A major obstacle to the use of adenovirus as a vector for gene therapy is the host immune response to hexon, the major protein component of the icosahedral capsid. A solution lies in creating novel vectors with modified or chimeric hexons to evade the immune response to native hexon. The crystal structure of hexon from human adenovirus type 5 (ad5), the type primarily used for gene therapy, has been determined to facilitate the design of such molecules. As the 951-amino-acid (aa) ad5 hexon sequence is closely related to that of ad2 (967 aa; 86% aa identity), the ad5 structure was solved by molecular replacement with a model constructed from the known ad2 hexon. During refinement, greater than 25% of the sequence was reassigned, resulting in a relocation of two epitope regions, from buried positions in the ad2 model to external sites at the top of the ad5 molecule. The resultant model is in better agreement with crystallographic data, while maintaining the overall topology of ad2 hexon. This work suggests that all hexons have the same basic fold and that the ad5 hexon structure provides an accurate and representative model for designing new adenovirus vectors.

Adenoviruses, Human↗
PubMed2005

Recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells.

Delivery of oligonucleotides (ON) into cells is a technical challenge. In this study, we utilized the capsid of the hepatitis B virus (HBV) to meet this goal. A single and short open reading frame of the virus programs efficient capsid production in bacteria. We show that these capsids can encapsulate ON in vitro and then mediate their delivery into cells with extreme efficiency. This process is cell type non-specific, rendering the recombinant HBV capsid a potentially valuable vehicle for ON delivery into a wide range of cultured cells.

Amino Acid Sequence↗
PubMed1996

A library of organic landscapes on filamentous phage.

A billion-clone library of filamentous phage with different surface structures ("landscapes') was generated by fusing random octapeptides to the N-terminus of all 4000 copies of the major coat protein. Such a "landscape library' might include clones exhibiting emergent properties that inhere in the entire surface architecture, not in the peptides by themselves. Because the diverse surface landscapes are displayed on viable phage, they can be surveyed for exceedingly rare functions using microbiological selection methods. Clones with several emergent properties of the sort envisioned were successfully selected, suggesting that landscape libraries have promise as a novel source of nanomaterials with exploitable surface properties.

Amino Acid Sequence↗
PubMed1992

The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells.

The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.

Animals↗
PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗
PubMed2006

Baculovirus display: a multifunctional technology for gene delivery and eukaryotic library development.

For over a decade, phage display has proven to be of immense value, allowing selection of a large variety of genes with novel functions from diverse libraries. However, the folding and modification requirements of complex proteins place a severe constraint on the type of protein that can be successfully displayed using this strategy, a restriction that could be resolved by similarly engineering a eukaryotic virus for display purposes. The quite recently established eukaryotic molecular biology tool, the baculovirus display vector system (BDVS), allows combination of genotype with phenotype and thereby enables presentation of eukaryotic proteins on the viral envelope or capsid. Data have shown that the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a versatile tool for eukaryotic virus display. Insertion of heterologous peptides and/or proteins into the viral surface by utilizing the major envelope glycoprotein gp64, or foreign membrane-derived counterparts, allows incorporation of the sequence of interest onto the surface of infected cells and virus particles. A number of strategies are being investigated in order to further develop the display capabilities of AcMNPV and improve the complexity of a library that may be accommodated. Numerous expression vectors for various approaches of surface display have already been developed. Further improvement of both insertion and selection strategies toward development of a refined tool for use in the creation of useful eukaryotic libraries is, however, needed. Here, the status of baculovirus display with respect to alteration of virus tropism, antigen presentation, transgene expression in mammalian cells, and development of eukaryotic libraries will be reviewed.

Animals↗
PubMed2000

Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity.

Collectins are important in the initial containment of a variety of pathogens, including influenza A virus (IAV). We provide the first systematic evaluation of the oligosaccharide-binding sites for pulmonary surfactant protein D (SP-D) on specific IAV coat glycoproteins and define the relationship between this binding and antiviral activity. With the use of several techniques, SP-D was found to bind via its carbohydrate-recognition domain (CRD) to mannosylated, N-linked carbohydrates on the HA(1) domain of the haemagglutinin (HA) and on the neuraminidase of IAV. Using a set of IAV strains that differed in the level and site of glycosylation, and a panel of recombinant collectins, we found that binding of SP-D to the globular domain of the HA was critical in mediating the inhibition of viral haemagglutination activity and infectivity. We also demonstrated that the pattern of binding of a collectin to IAV glycoproteins can be modified by altering the monosaccharide-binding affinity of its CRD or by linking the CRD to a different N-terminal/collagen domain. These studies clarify the mechanisms of viral neutralization by collectins and might be useful in engineering collectins for enhanced antiviral activity.

Amidohydrolases↗
PubMed2005

HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo.

Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

Animals↗
PubMed1986

Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region.

All picornaviral peptides are derived by progressive posttranslational cleavage of a giant precursor polyprotein. Translation of encephalomyocarditis virus (EMC) RNA in rabbit reticulocyte extracts produces active viral peptides, including protease 3C, which is responsible for many cleavage reactions within the processing cascade. DNA plasmids containing 5' noncoding sequences of EMC linked to other portions of the viral genome were constructed and transcribed into RNA. Like virion RNA, the clone-derived transcripts directed efficient protein translation in vitro. The 5'-linked constructions may represent examples of a general method for cell-free expression of any cloned gene segment. One construction produced a self-cleaving P3 region precursor, which contained active 3C protease. A genetically engineered insertion within the 3C sequences eliminated endogenous self-cleavage activity without altering the ability of the P3 peptide to serve as substrate in bimolecular reactions with added 3C. Another plasmid encoding the L-VP0 portion of the capsid region was used to demonstrate that scission between the leader peptide (L) and capsid protein VP0 can be catalyzed by 3C. The enzyme responsible for this step was previously unidentified. A rapid purification scheme for isolation of 3C from EMC-infected HeLa cells is also presented.

Base Sequence↗
PubMed2004

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI.

To explore the potential application of capsid-targeted viral inactivation (CTVI) strategy in prophylactic model against dengue virus (DV) infection, here we fused a Ca(2+)-dependent nuclease, staphylococcal nuclease (SN), to the capsid protein of dengue 2 virus (D2C) at the carboxyl terminal, and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C. A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5mgr;/ml blasticidin. The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays. The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay. It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained. The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells. Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

Amino Acid Sequence↗
PubMed1999

Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.

Abatacept↗
PubMed2005

Adenovirus-mediated transgene-engineered dendritic cell vaccine of cancer.

Dendritic cells (DCs) are the most effective antigen presenting cells (APCs) to elicit both primary and secondary T-cell response that is critical for antitumor immunity and elimination of intracellular pathogens. Therefore, DCs pulsed ex vivo with antigens have the potential used as cell-based vaccines against tumors. Viral vectors derived from adenoviruses have been extensively used to pulse DCs ex vivo by delivering genes encoding immunomodulatory molecules and tumor antigens to DCs since these vectors are relatively safe, effective in inducing the maturation of DCs, and can accommodate large expression cassettes encoding antigens. One of the hurdles for gene delivery to DCs by adenovirus (Ad) vectors, however, is low transfection efficiency of DCs due to the paucity of Ad receptor on DCs. To overcome this obstacle, targeted Ad vectors have been made by modifying viral capsid proteins. These targeted Ad vectors not only enhance the gene delivery to DCs, but also allow in vivo gene delivery to DCs, thus avoiding ex vivo manipulation of DCs.

Adenoviridae↗