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.

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 Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.

1981 ◽  
Vol 1 (9) ◽  
pp. 807-813 ◽  
Author(s):  
M G Katze ◽  
H Persson ◽  
L Philipson
Keyword(s):  
Gene Product ◽  
Messenger Rna ◽  
Adenovirus Type ◽  
Negative Regulator ◽  
Early Gene ◽  
In Vitro Translation ◽  
Mapping Technique ◽  
Cellular Gene ◽  
Infected Cells ◽  
Adenovirus Type 5

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

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 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 An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

2001 ◽  
Vol 75 (14) ◽  
pp. 6303-6309 ◽  
Author(s):  
Marintha L. Heil ◽  
Alison Albee ◽  
James H. Strauss ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Heparan Sulfate ◽  
Amino Acid Substitution ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Ross River Virus ◽  
Wild Type ◽  
Coding Region ◽  
Sulfate Production ◽  
E2 Glycoprotein

ABSTRACT Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.

Host-range mutants of adenovirus type 5 defective for growth in HeLa cells

Virology ◽  
1977 ◽  
Vol 77 (1) ◽  
pp. 319-329 ◽  
Author(s):  
Tim Harrison ◽  
Frank Graham ◽  
Jim Williams
Keyword(s):  
Host Range ◽  
Hela Cells ◽  
Adenovirus Type ◽  
Adenovirus Type 5

A dominant temperature-sensitive assembly mutant of adenovirus type 2

1979 ◽  
Vol 25 (5) ◽  
pp. 646-649 ◽  
Author(s):  
Eric B. Carstens ◽  
Johanne Magnan ◽  
Joseph Weber
Keyword(s):  
Negative Temperature ◽  
Adenovirus Type ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Sensitive Mutant ◽  
Virion Assembly ◽  
Temperature Sensitive Mutant

An assembly negative temperature-sensitive mutant of Ad2, ts48 was shown to exert dominance over other ts mutants and wild-type virus during coinfection, by inhibiting virion assembly. Dominance was only expressed at the nonpermissive temperature.

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.

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