Baculovirus GP64-mediated entry into mammalian cells

Area of Science:

• Virology
• Cell Biology
• Gene Therapy

Background:

• Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an efficient viral vector for insect and mammalian cells.
• Lentivirus vectors pseudotyped with baculovirus GP64 show enhanced transduction compared to VSV-G.
• Mechanisms of AcMNPV and GP64-pseudotyped virus entry into mammalian cells are not fully understood.

Purpose of the Study:

• To elucidate the entry mechanisms of AcMNPV and GP64-bearing pseudotyped viruses into mammalian cells.
• To identify key cellular factors involved in baculovirus GP64-mediated viral entry.
• To inform the development of more efficient and specific viral vectors for gene delivery.

Main Methods:

• Generated pseudotyped VSV vectors (NP NPVpv, VSVpv, MLVpv) with baculovirus, VSV, and MLV envelope proteins.
• Used methyl-β-cyclodextrin to deplete cellular membrane cholesterol.
• Employed inhibitors and dominant-negative mutants for dynamin, clathrin, and caveolin-mediated endocytosis.
• Assessed the impact of macropinocytosis inhibition on viral internalization.

Main Results:

• Cholesterol depletion inhibited GP64-mediated internalization but not cell surface attachment.
• Dynamin- and clathrin-dependent endocytosis pathways were essential for AcMNPV and NPVpv internalization.
• Caveolin-mediated endocytosis was not involved in the entry process.
• Macropinocytosis inhibition reduced GP64-mediated internalization.

Conclusions:

• Cholesterol, dynamin- and clathrin-dependent endocytosis, and macropinocytosis are critical for baculovirus GP64 entry into mammalian cells.
• Understanding these mechanisms is vital for optimizing baculovirus-based viral vectors for gene therapy applications.