Genome stability of murine cytomegalovirus vectors with large deletions and insertions

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 26-18% of the wild type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Next, two independent stuffer DNAs were inserted into the Q4 BAC resulting in Q4-LAD and Q4-LRBAs BACs. LAD is an AT-rich DNA sequence based on inactivated human adenovirus genome (34 kbp) and a non-coding portion of human LRBA (LPS responsive beige-like anchor protein) gene a non-coding portion of human LRBA gene which is GC-rich (36 kbp). The Q4-LRBAs BAC was further modified by inserting two different transgene expression cassettes encoding for either Gaussian luciferase (GLuc) resulting in Q4-LRBAs-GLuc. First, we rescued replicating vectors after transfection of MEFs with the above described BACs. Each vector preparation was passaged up to 10 passage on MEFs. Then MEF cells (70% confluence) were infected with Q4, Q4-LAD, and Q4-LRBAs-GLuc at MOI 0.5. derived from passage 1, 5 and 10. As control we used lysates of wild type BAC derived MCMV infected MEFs after passages 1, 5, 10, 15, and 20. After 48 hpi, virus particles were harvested, purified, and DNA was extracted using the NucleoSpin Tissue kit (Macherey-Nagel, Germany). Illumina NGS was performed on 100 ng DNA template for each sample (paired end sequencing, 2×150 bp, 5 Mio reads/sample). This dataset contains the raw sequencing data (individually named .zip files) and a table (an .xlsx file packed into MCMV Vector Stability_VACCINES_2024.zip) with the detailed specifications of the dataset. The reference sequences, which we used for the analysis in our Vaccines paper (Riedl et al. Vaccines, 2024), are provided in a zipped folder in .gb format.