CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts.
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Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdDeltaRGDbetagal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alphaV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)betagal (bearing seven lysines on the end of fiber) (b) AdF(RGD)betagal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK betagal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors. (+info)
The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.
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The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface. (+info)
Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.
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The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus. (+info)
Time-resolved fluorescence investigation of the human immunodeficiency virus type 1 nucleocapsid protein: influence of the binding of nucleic acids.
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Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar. (+info)
Identification of additional genes that influence baculovirus late gene expression.
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We were unable to confirm transient late gene expression using constructs of 18 genes that had been reported to support Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) late gene expression when transfected into Spodoptera frugiperda cells [Lu, A., and Miller, L. K. (1995). J. Virol. 69, 975-982]. Three genes (orf66, orf68, and orf41) were included, all or in part, in the constructs used in that study, but they had not been independently tested. Therefore we investigated these and neighboring orfs for their influence on late gene expression. We found that orf41 was required for late gene expression and that sequences within orf45 appeared to be required for the expression of orf41. Although orf66 and orf68 did not appear to affect late gene expression, orf69 stimulated expression. orf69 was found to have high homology to recent entries in GenBank from a variety of organisms. In addition, it was found that orf121, which was shown to be involved in early gene expression, and the viral homolog of pcna did not influence late gene expression. (+info)
A new picornavirus isolated from bank voles (Clethrionomys glareolus).
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A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype. (+info)
Direct evidence that the proton motive force inhibits membrane translocation of positively charged residues within membrane proteins.
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The M13 phage procoat protein requires both its signal sequence and its membrane anchor sequence in the mature part of the protein for membrane insertion. Translocation of its short acidic periplasmic loop is stimulated by the proton motive force (pmf) and does not require the Sec components. We now find that the pmf becomes increasingly important for the translocation of negatively charged residues within procoat when the hydrophobicity of the signal or membrane anchor is incrementally reduced. In contrast, we find that the pmf inhibits translocation of the periplasmic loop when it contains one or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compromised. No pmf effect is observed for translocation of an uncharged periplasmic loop even when the hydrophobicity is reduced. We also show that the Delta Psi component of the pmf is necessary and sufficient for insertion of representative constructs and that the translocation effects of charged residues are primarily due to the DeltaPsi component of the pmf and not the pH component. (+info)
Interactions of heterologous DNA with polyomavirus major structural protein, VP1.
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'Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstova et al. (1995) Hum. Gene Ther. 6, 297-3061. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain. (+info)