Self-Repairing Herpesvirus Saimiri Deletion Variants

Abstract

Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12-14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells.

Keywords: herpesvirus saimiri; recombination; vector.

Figure 1

Scheme of the two separate recombination events which constitute the viral self-repair of herpesvirus saimiri (HVS) virus variants within permissive epithelial cells. In the upper panel, WT22fDR carries direct repeats of ORF22 while in the middle panel, WT29fDX carries inverted repeats of ORF29b (indicated by color gradients). The primer combinations used to monitor viral self-repair by PCR are indicated (F: forward primer, R: reverse primer). In the lowest panel, the self-repaired virus genome is shown without F plasmid vector moiety (miniF) sequence (orange-colored genes). The resulting DNA is free of residual base pairs and the ORFs previously harboring the miniF are restored to their original state. Images taken and modified from [27].

Figure 2

Genomic monitoring and replication properties of the self-repairing virus variants WT22fDR and WT29fDX. (a) PCR analysis of viral genomes during passaging of the virus variants in permissive OMK cells. ORF75 served as a viral control, sopA and cat were parameters for the respective part of the miniF that was to be lost, and ORF22/29 PCR indicated self-restoration of the ORF harboring the miniF sequence. Inverse gray shades are depicted. All band sizes were of the expected size. (b) Exemplary replication kinetics of one experiment of at least three separate experiments of the self-repaired virus variants after passage 9 (p9). The harvested supernatants were titrated in three individual dilution series of which mean and standard deviation are indicated. Virus titers are given in plaque-forming units (pfu)/mL in relation to the day they were harvested.

Figure 3

Restriction-fragment length polymorphism analysis (RFLP) of isolated HVS C488 variant DNA subjected to restriction digestion with enzymes EcoRV and NcoI, respectively. The DNA fragments were separated on a 0.7% agarose gel. Gray shades are displayed inversely. (a) BAC DNA of the varying wild-type-like virus variants with miniF located in ORF14 (WTf), ORF22 (WT22fDR) and ORF29 (WT29fDX), respectively. (b) Viral DNA isolated after nine passages in OMK cells compared to the wild-type virus HVS C488. Self-repair was reliably completed at that passage and the miniF sequence removed from the viral genome.

Figure 4

Self-repair of deletion variants and replication kinetics in self-repaired state. (a) Conventional PCR after virus passage (p) 1 to 7. ORF75 PCR served as viral control, sopA and cat as indicators for the self-repair, as well as the PCR for ORF22 and ORF29, respectively. For the variant WT29fDX∆3∆M21, deletions of ORF3 and M21 were proven. WT29fDXHTLV was checked by sequencing instead after passage 9 and was found to be correct. All bands were of the expected size, indicated in kilobase pairs (kb). Gray shades are depicted in inverted mode. (b) Replication kinetics of the different deletion variants are plotted compared to the wild-type virus or the self-repaired wild-type parental virus variant. Mean titers and standard deviation are indicated.

Figure 5

Scatter-plots of FITC-positive cells in percent indicating annexin V-presenting cells. (a) Percent of apoptotic cells in transformed cells of donors K38 and K39. Self-repaired wild-type virus WT(29fDX) or a deletion variant with either ORF16 or ORF71 deleted were initially used to transform the cells 31 months prior. Effects of apoptosis inducers hydrogen peroxide (H₂O₂) or camptothecin at indicated final concentrations were investigated 4 h and 24 h post-induction. (b) Effects of the two inductors compared between cells of the same donor, transformed with the different self-repaired virus variants 24 h post-induction. Cell numbers did not suffice for more than one (K38 cell lines) and two experiments (K39 cell lines), respectively.