Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase

Cristina Capella, Michael John Beltejar, Caitlin Brown, Vincent Fong, Waaqo Daddacha, Baek Kim, Stephen Dewhurst

Research output: Contribution to journalArticle

Abstract

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1DNApolymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We there fore tested whether mutations in regions of the adenovirus type 5 (Ad5)DNApolymerase that interact with the dNTP substrate orDNAtemplate could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viralDNApolymerase utilizes dNTP substrates.

Original languageEnglish (US)
Pages (from-to)10484-10493
Number of pages10
JournalJournal of Virology
Volume86
Issue number19
DOIs
StatePublished - Oct 1 2012
Externally publishedYes

Fingerprint

DNA-directed DNA polymerase
Adenoviridae
DNA-Directed DNA Polymerase
mutagenesis
Mutagenesis
virus replication
Virus Replication
mutation
viruses
cells
lungs
Mutation
Viruses
Lung
cell lines
mutants
Cell Line
Neoplasms
triphosphoric acid
carcinoma

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase. / Capella, Cristina; Beltejar, Michael John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen.

In: Journal of Virology, Vol. 86, No. 19, 01.10.2012, p. 10484-10493.

Research output: Contribution to journalArticle

Capella, Cristina ; Beltejar, Michael John ; Brown, Caitlin ; Fong, Vincent ; Daddacha, Waaqo ; Kim, Baek ; Dewhurst, Stephen. / Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase. In: Journal of Virology. 2012 ; Vol. 86, No. 19. pp. 10484-10493.
@article{824252afa8b44a9880f8c0ca3c153952,
title = "Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase",
abstract = "Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1DNApolymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We there fore tested whether mutations in regions of the adenovirus type 5 (Ad5)DNApolymerase that interact with the dNTP substrate orDNAtemplate could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viralDNApolymerase utilizes dNTP substrates.",
author = "Cristina Capella and Beltejar, {Michael John} and Caitlin Brown and Vincent Fong and Waaqo Daddacha and Baek Kim and Stephen Dewhurst",
year = "2012",
month = "10",
day = "1",
doi = "10.1128/JVI.00739-12",
language = "English (US)",
volume = "86",
pages = "10484--10493",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "19",

}

TY - JOUR

T1 - Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase

AU - Capella, Cristina

AU - Beltejar, Michael John

AU - Brown, Caitlin

AU - Fong, Vincent

AU - Daddacha, Waaqo

AU - Kim, Baek

AU - Dewhurst, Stephen

PY - 2012/10/1

Y1 - 2012/10/1

N2 - Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1DNApolymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We there fore tested whether mutations in regions of the adenovirus type 5 (Ad5)DNApolymerase that interact with the dNTP substrate orDNAtemplate could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viralDNApolymerase utilizes dNTP substrates.

AB - Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1DNApolymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We there fore tested whether mutations in regions of the adenovirus type 5 (Ad5)DNApolymerase that interact with the dNTP substrate orDNAtemplate could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viralDNApolymerase utilizes dNTP substrates.

UR - http://www.scopus.com/inward/record.url?scp=84869020077&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84869020077&partnerID=8YFLogxK

U2 - 10.1128/JVI.00739-12

DO - 10.1128/JVI.00739-12

M3 - Article

C2 - 22811532

AN - SCOPUS:84869020077

VL - 86

SP - 10484

EP - 10493

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 19

ER -