Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome

1984 ◽  
Vol 4 (2) ◽  
pp. 302-309
Author(s):  
D Hanahan ◽  
Y Gluzman
Keyword(s):  
Viral Genome ◽  
Infectious Virus ◽  
Adenovirus Type ◽  
Small Fragment ◽  
Origin Of Replication ◽  
Wild Type ◽  
Base Pairs ◽  
Bacterial Plasmid ◽  
Dna Fragment ◽  
Adenovirus Type 5

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

scholarly journals Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

1984 ◽  
Vol 4 (2) ◽  
pp. 302-309 ◽  
Author(s):  
D Hanahan ◽  
Y Gluzman
Keyword(s):  
Viral Genome ◽  
Infectious Virus ◽  
Adenovirus Type ◽  
Small Fragment ◽  
Origin Of Replication ◽  
Wild Type ◽  
Base Pairs ◽  
Bacterial Plasmid ◽  
Dna Fragment ◽  
Adenovirus Type 5

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region

1982 ◽  
Vol 152 (2) ◽  
pp. 829-839
Author(s):  
A M Easton ◽  
R H Rownd
Keyword(s):  
Gene Expression ◽  
Target Site ◽  
Plasmid Replication ◽  
Lacz Gene ◽  
Origin Of Replication ◽  
Wild Type ◽  
Base Pairs ◽  
Replication Region ◽  
Lambda Phages ◽  
Beta Galactosidase

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.

A study of possible biohazards in the fluorescent antibody test using adenovirus, coxsackievirus, herpesvirus, and respiratory syncytial virus as antigens

1976 ◽  
Vol 4 (4) ◽  
pp. 322-325
Author(s):  
D Bardell
Keyword(s):  
Respiratory Syncytial Virus ◽  
Infectious Virus ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Adenovirus Type ◽  
Antigenic Determinants ◽  
Syncytial Virus ◽  
Simplex Virus ◽  
Adenovirus Type 5

Infectious adenovirus type 5 and coxsackievirus type B5, both nonlipid-containing viruses, were isolated from cells fixed in acetone at 22 degrees C for 15 min, from acetone used for fixation, from the solution used for washing slides during the fluorescent antibody procedure, and after complete processing of antigen preparations with serial twofold dilutions of human antisera and fluorescein-labeled goat anti-human immunoglobulin G. Lipid-containing herpes simplex virus type 1 and respiratory syncytial virus were inactivated by acetone, and infectious virus could not be recovered at any stage in the fluorescent antibody test. Fixation in acetone at 56 degrees C destroyed the infectivity of adenovirus 5 and coxsackievirus B5 within 30 min, but no adverse effect on the antigenic determinants of either virus occurred until after 60 min, thus demonstrating that these antigens can be utilized without the hazard of infectious virus.

scholarly journals Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes

2002 ◽  
Vol 83 (6) ◽  
pp. 1311-1314 ◽  
Author(s):  
Hendrik J. Rademaker ◽  
Mohamed A. Abou El Hassan ◽  
Gijs A. Versteeg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Hepg2 Cells ◽  
Adenovirus Type ◽  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Wild Type ◽  
Genome Packaging ◽  
Firefly Luciferase Gene ◽  
Adenovirus Vectors ◽  
Initiation Of Dna Replication ◽  
Adenovirus Type 5

Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a ΔE1ΔE3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing ΔE1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.

scholarly journals Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

2001 ◽  
Vol 75 (23) ◽  
pp. 11284-11291 ◽  
Author(s):  
David A. Einfeld ◽  
Rosanna Schroeder ◽  
Peter W. Roelvink ◽  
Alena Lizonova ◽  
C. Richter King ◽  
...  
Keyword(s):  
Adenovirus Type ◽  
Wild Type ◽  
Base Modification ◽  
Model Following ◽  
Vector Distribution ◽  
High Level ◽  
Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

scholarly journals The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

1986 ◽  
Vol 6 (2) ◽  
pp. 470-476 ◽  
Author(s):  
S Pilder ◽  
M Moore ◽  
J Logan ◽  
T Shenk
Keyword(s):  
Hela Cells ◽  
Adenovirus Type ◽  
Transcription Unit ◽  
Early Gene ◽  
Wild Type Virus ◽  
Wild Type ◽  
Coding Region ◽  
Viral Mrnas ◽  
Cellular Mrnas ◽  
Adenovirus Type 5

The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

scholarly journals An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

1999 ◽  
Vol 73 (6) ◽  
pp. 4600-4610 ◽  
Author(s):  
Joseph S. Orlando ◽  
David A. Ornelles
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Protein Function ◽  
Alpha Helix ◽  
Adenovirus Type ◽  
Wild Type ◽  
Reading Frame ◽  
Α Helix ◽  
Amphipathic Alpha Helix ◽  
Adenovirus Type 5

ABSTRACT A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

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