A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes

Abstract

Advances in DNA transposition technology have recently generated efficient tools for various types of functional genetic analyses. We demonstrate here the power of the bacteriophage Mu-derived in vitro DNA transposition system for modification and functional characterization of a complete bacterial virus genome. The linear double-stranded DNA genome of Escherichia coli bacteriophage PRD1 was studied by insertion mutagenesis with reporter mini-Mu transposons that were integrated in vitro into isolated genomic DNA. After introduction into bacterial cells by electroporation, recombinant transposon-containing virus clones were identified by autoradiography or visual blue-white screening employing ␣-complementation of E. coli ␤-galactosidase. Additionally, a modified transposon with engineered NotI sites at both ends was used to introduce novel restriction sites into the phage genome. Analysis of the transposon integration sites in the genomes of viable recombinant phage generated a functional map, collectively indicating genes and genomic regions essential and nonessential for virus propagation. Moreover, promoterless transposons defined the direction of transcription within several insert-tolerant genomic regions. These strategies for the analysis of viral genomes are of a general nature and therefore may be applied to functional genomics studies in all prokaryotic and eukaryotic cell viruses.

Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

“…However, these methods can produce variable or biased insertions, and library generation efficiencies are often too low to apply to larger genes. Transposon-based insertional mutagenesis has recently emerged as an efficient means of studying the features of viral genomes (1,5,22,25,44). We hypothesized that a transposonbased approach for saturation insertional mutagenesis, coupled with a high-throughput viral-based library selection process, could rapidly identify optimal sites within VSV-G that could functionally incorporate a novel peptide sequence.…”

Genome Characterization of Lipid-Containing Marine Bacteriophage PM2 by Transposon Insertion Mutagenesis

“…strain ER72M2 cells were cultured in SB broth or on SB agar plates (33) at 28°C. Escherichia coli K-12 DH5␣ (Life Technologies) was used for transposon preparation as described previously (20,50).…”

“…One of the most versatile methods is based on the bacteriophage Mu in vitro DNA transposition reaction, which can be used to disseminate custom-designed mini-Mu transposons into any DNA, including viral genomes. This strategy has been used successfully for the functional characterization of linear dsDNA genomes of bacteriophages PRD1 (50) and YeO3-12 (31). A related strategy employing transposon ends has been applied to human immunodeficiency virus (34) and potato virus A (30) RNA genome characterization.…”

“…Recently, we have initiated a structural characterization of phage PM2 as a model phage system (2,26). Since no genetic tools are available in this system, we set out to probe the PM2 genome using the phage Mu-derived in vitro transposition mutagenesis technology, which represents an efficient alternative strategy for functional genome characterization (50).…”

“…Transposon mutagenesis can be used to map essential versus nonessential regions in viral genomes (8,48,50,54). One of the most versatile methods is based on the bacteriophage Mu in vitro DNA transposition reaction, which can be used to disseminate custom-designed mini-Mu transposons into any DNA, including viral genomes.…”