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Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells
Published on: November 12, 2015
Functional genetic and biophysical analyses of membrane disruption by human adenovirus
Crystal L Moyer1, Christopher M Wiethoff, Oana Maier
1Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.
Insights
Adenovirus (AdV) protein VI is identified as the key factor for penetrating endosomes during infection. This study provides direct evidence and reveals the biochemical mechanism behind its membrane interactions.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Adenovirus (AdV) infection requires endosome penetration.
- The internal capsid protein VI was previously implicated in AdV membrane lysis.
- Direct evidence for protein VI's role in endosome penetration during cell entry was lacking.
Purpose of the Study:
- To definitively identify the AdV protein responsible for endosome penetration.
- To investigate the role of protein VI's N-terminal amphipathic α-helix in membrane lysis.
- To elucidate the biochemical basis of protein VI's membrane interactions.
Main Methods:
- Engineered random mutations in protein VI's N-terminal α-helix within the AdV genome.
- Utilized a novel technique combining error-prone PCR and recombineering.
- Performed biophysical analysis of mutated protein VI's membrane insertion.
Main Results:
- Identified a specific L40Q mutation in protein VI that significantly reduced AdV infectivity.
- Demonstrated that the L40Q mutation selectively impaired endosome penetration.
- Showed reduced membrane insertion of VI-L40Q, correlating with impaired endosomalysis.
Conclusions:
- Protein VI is the critical membrane lytic factor for AdV during cellular entry.
- The N-terminal amphipathic α-helix of protein VI is crucial for its membrane lytic activity.
- The L40Q mutation provides insight into the biochemical mechanisms of AdV membrane interaction.
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