Human cytomegalovirus (HCMV) is a widely spread β-herpesvirus that establishes life-long latency after primary infection. In healthy individuals, primary infection is usually subclinical. However, primary infection or reactivation of HCMV in immunosuppressed patients may cause severe disease, eventually with fatal outcome. A common cellular defense mechanism to prevent virus replication is to induce apoptosis, as viruses depend on the host cell for replication. However, CMV is one of many viruses that have evolved genes encoding anti-apoptotic proteins. The human cytomegalovirus UL37 exon 1 (UL37x1) protein prevents release of cytochrome C from mitochondria and thereby inhibits apoptosis. The protein is also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA).
AD169 is a wild-type strain of human CMV that is commonly used in the laboratory. Its entire nucleotide sequence is known. The AD169ΔUL37x1 deletion mutant was made by replacing the UL37x1 gene in the AD169-BAC clone with a kanamycin selection cassette.
The ΔUL37x1 deletion mutant was constructed by replacing the UL37x1 gene in AD169-BAC (NCBI accession ID 146999) with a kanamycin selection cassette (kanR) using DH10B Escherichia coli cells expressing the recombination functions red α/β/χ as described previously (Datsenko and Wanner, 2000 and Hahn et al., 2004). Viral sequences replaced by kanR extended from nt 52293 to 52713. BAC plasmid was isolated from the bacteria, and the mutant ΔUL37x1 BAC DNA was verified by sequencing before transfection and reconstitution of recombinant virus. Briefly, 3 × 106 MRC-5 cells were resuspended in media and mixed with viral pADCRE-ΔUL37x1 DNA, pCMV71 (Liu and Stinski, 1992) and pCMV-E1B, electroporated and cultured for approximately seven to 10 days. The supernatant was harvested and used for preparation of a highly purified virus stock.
