ABSTRACT
Bacterial artificial chromosomes (BACs) were constructed containing full-length, infectious DNA of HSV-1 strains 17 and KOS. To generate BACs without altering viral genes, sequences required for selection and propagation of the BAC were placed between the U(L)37 and U(L)38 genes, and flanked by LoxP sites. The system was tested by studying multiple properties of these HSV-1 BAC constructs in vitro and in vivo following propagation in bacteria, virus reconstitution from HSV-BAC DNA in eukaryotic cells, and Cre-recombinase-mediated excision of the BAC backbone. Based on in vitro growth in mouse embryo fibroblasts and in vivo growth in mouse corneas and trigeminal ganglia, the strain KOS BAC-derived virus behaved similarly to wild-type. Small changes in neurovirulence were, however, observed. The strain 17 BAC-derived virus exhibited modest decreases in growth and virulence compared to wild-type. Modest differences were observed in reactivation from latency with both strain KOS and 17 BAC-derived viruses. In addition, the system was further validated by performing mutagenesis of the BACs by allelic exchange in E. coli. These BACs are suitable for the rapid generation of recombinant viruses for pathogenesis and other studies, but as with all mutagenesis systems, care must be taken in their construction and repair.
Subject(s)
Capsid Proteins/genetics , Chromosomes, Artificial, Bacterial , Herpesvirus 1, Human/genetics , Viral Structural Proteins/genetics , Animals , Chlorocebus aethiops , Escherichia coli/genetics , Female , Genome, Viral , Herpesvirus 1, Human/physiology , Mice , Mutagenesis , Vero Cells , Virus Activation , Virus ReplicationABSTRACT
The virion host shutoff protein (Vhs) of herpes simplex virus type 1 induces destabilization of mRNA following infection. Our study of primary neurons from CD-1 mice demonstrates that vhs is functional in neurons but that more Vhs is required to mediate RNA degradation in neurons than in other susceptible cells.
