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Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein
K A Solomon1, A K Robbins, L W Enquist
1Viral Diseases Research, Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328.
Abstract:
The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.
Insights
Pseudorabies virus gIII protein's membrane anchor is crucial for virion localization. Unexpectedly, its specific sequence isn't vital, suggesting protein conformation influences processing and incorporation.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Pseudorabies virus (PRV) is an important pathogen affecting swine.
- The gIII protein is a PRV envelope glycoprotein involved in virus assembly.
- Understanding glycoprotein anchoring and localization is key to viral pathogenesis.
Purpose of the Study:
- To genetically map the functional membrane anchor of the PRV gIII protein.
- To investigate the role of the hydrophobic domain in gIII membrane retention and virion localization.
- To elucidate the mechanism of glycoprotein incorporation into PRV virions.
Main Methods:
- Site-directed mutagenesis to create mutant gIII proteins.
- Analysis of gIII protein association with virus particles.
- Assessment of gIII membrane retention, export, and posttranslational processing.
Main Results:
- The functional gIII membrane anchor was localized between amino acids 443 and 466.
- Mutant gIII proteins lacking the membrane anchor were not found in virus particles.
- The specific hydrophobic sequence was replaceable with leucine without affecting membrane retention or localization.
- Mutations within the hydrophobic region impacted gIII processing efficiency and virion localization.
Conclusions:
- Membrane retention is essential for gIII virion localization.
- The hydrophobic domain's composition, not its specific sequence, influences gIII conformation, processing, and export.
- Virion localization is likely an indirect consequence of efficient gIII processing.