Downstream Sequences Control the Processing of the Pestivirus E^rns-E1 Precursor.

Area of Science:

• Virology
• Molecular Biology
• Protein Processing

Background:

• Enveloped viruses, including pestiviruses, rely on cellular proteases for structural protein maturation.
• Signal peptidase (SPase) plays a critical role in cleaving viral glycoproteins.
• Pestiviruses exhibit unusual SPase cleavage sites, particularly at the E^rns-E1 junction.

Purpose of the Study:

• To investigate the role of the E1 protein sequence in the signal peptidase-mediated cleavage of the pestiviral E^rns-E1 precursor.
• To understand the regulation and order of processing events for pestiviral glycoproteins.
• To elucidate the impact of E1 sequence integrity on viral protein maturation and secretion.

Main Methods:

• Site-directed mutagenesis to create carboxy-terminal truncations and internal deletions in the E1 protein.
• Analysis of protein processing efficiency using Western blotting and secretion assays.
• Investigation of the effect of mutations in the von Heijne sequence on cleavage events.

Main Results:

• Cleavage at the E^rns-E1 site is inefficient with truncated or internally deleted E1 sequences, reducing processing to <30% of wild-type levels.
• Carboxy-terminal truncation of E1 by >30 amino acids results in significant secretion of uncleaved fusion proteins.
• Mutations in the von Heijne sequence upstream of E2 disrupt both E1/E2 and E^rns/E1 cleavage, indicating ordered processing.
• E^rns-E1 cleavage is dependent on the prior cleavage at the E1/E2 site.

Conclusions:

• The integrity of the E1 protein sequence is essential for efficient signal peptidase-mediated cleavage of the E^rns-E1 precursor in pestiviruses.
• Impaired processing due to E1 truncation leads to increased secretion of uncleaved viral proteins.
• Pestiviral glycoprotein processing by SPase occurs in a highly regulated, ordered manner, dependent on specific sequence elements and cleavage events.