Generation and characterization of a Cowpox virus mutant lacking host range factor CP77

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Cowpox virus (CPXV) host range factor CP77 is known to be required for viral replication in Chinese hamster ovary (CHO) cells.
• The precise molecular mechanisms by which CP77 influences viral host range remain largely uncharacterized.

Purpose of the Study:

• To investigate the role of CP77 in CPXV replication and host range specificity.
• To construct and characterize a CPXVΔCP77 deletion mutant to elucidate CP77 function.

Main Methods:

• Bacterial artificial chromosome (BAC) technology was used to create a CPXVΔCP77 deletion mutant.
• Whole genome sequencing confirmed the integrity of the BAC-derived viral DNA.
• In vitro replication assays were performed in various cell lines (HEK293T, CHO-K1) and in ovo.

Main Results:

• CPXVΔCP77 replication was significantly attenuated in CHO-K1 cells, but not in HEK293T cells.
• The replication defect in CHO cells was associated with a lack of late viral protein expression.
• Recombinant expression of CP77 restored replication in CHO cells, confirming a CP77-dependent host range effect.
• This effect was observed in both CPXV and Camelpox virus backgrounds.
• No significant differences in replication or cytopathic effects were observed in other tested cell types, including other hamster-derived cells.

Conclusions:

• CP77 mediates a specific host range effect in CHO cells, crucial for efficient CPXV replication.
• The precise function of CP77 beyond this host range effect requires further investigation.
• The generated vCPXVΔCP77 deletion mutant serves as a valuable tool for future research on CP77's functions.