Directed adenovirus evolution using engineered mutator viral polymerases.
Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad's intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, 'accelerated-evolution' approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad's intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands.
Taco G
TG
Uil
Jort
J
Vellinga
Jeroen
J
de Vrij
Sanne K
SK
van den Hengel
Martijn J W E
MJ
Rabelink
Steve J
SJ
Cramer
Julia J M
JJ
Eekels
Yavuz
Y
Ariyurek
Michiel
M
van Galen
Rob C
RC
Hoeben
D000256:Adenoviridae / Q000201:enzymology / Q000235:genetics*; D017503:Adenovirus E3 Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D045744:Cell Line, Tumor; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D050504:Oncolytic Viruses / Q000235:genetics*; D012326:RNA Splicing; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
D017503:Adenovirus E3 Proteins; D014764:Viral Proteins; C099609:adenovirus death protein, Adenovirus; D004259:DNA-Directed DNA Polymerase