DNA synthesis in chromatin preparations from human fibroblasts infected by cytomegalovirus

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Human cytomegalovirus (HCMV) is a significant pathogen, particularly in immunocompromised individuals.
• Understanding HCMV replication mechanisms is crucial for developing antiviral therapies.
• Viral DNA synthesis is a key target for inhibiting viral replication.

Purpose of the Study:

• To investigate the characteristics of endogenous DNA synthesis in HCMV-infected human fibroblasts.
• To determine whether viral or host cell DNA polymerases are responsible for DNA incorporation.
• To elucidate the role of viral DNA polymerase in HCMV replication.

Main Methods:

• Chromatin preparation from (14C)-thymidine pulse-labeled HCMV-infected human fibroblasts.
• Assay for endogenous (3H)-thymidine triphosphate incorporation.
• Isopycnic centrifugation of doubly labeled DNA in CsCl gradients.
• Density labeling experiments using bromodeoxyuridine triphosphate.
• Analysis of viral DNA polymerase activity in infected cell chromatin.

Main Results:

• Endogenous DNA synthesis activity was detected in chromatin from HCMV-infected fibroblasts.
• This activity was relatively salt-resistant and sensitive to phosphonoacetic acid, an inhibitor of viral DNA polymerase.
• Isopycnic centrifugation showed incorporation into both viral and host cell DNA.
• Density labeling indicated that incorporation into viral DNA was due to replicative DNA synthesis.
• Chromatin contained both cellular and viral DNA polymerase activities.

Conclusions:

• HCMV-infected human fibroblasts possess endogenous DNA synthesis capabilities.
• The observed DNA incorporation is primarily associated with viral DNA replication, driven by viral DNA polymerase.
• These findings provide insights into the molecular mechanisms of HCMV DNA replication and potential therapeutic targets.