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The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

Zarate-Perez F, Bardelli M, Burgner JW, Villamil-Jarauta M, Das K, Kekilli D, Mansilla-Soto J, Linden RM, Escalante CR - PLoS Pathog. (2012)

Bottom Line: We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers.We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases.Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America.

ABSTRACT
The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

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Related in: MedlinePlus

Effect of Y224 mutation on AAV-2 virus liability.Comparison of the production of rAAV2-GFP infectious particles in presence of wt (squares) or Y224A Rep (triangles). rAAV2-GFP particles were produced in 293T cells in presence of wt or Y224A Rep. Varying volumes of crude lysate (in µl, x-axis) were added to HeLa cells and the percentage GFP positive -infected- cells was determined by FACS analysis. Data from four experiments are represented as mean ± s.e.m.
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ppat-1002764-g009: Effect of Y224 mutation on AAV-2 virus liability.Comparison of the production of rAAV2-GFP infectious particles in presence of wt (squares) or Y224A Rep (triangles). rAAV2-GFP particles were produced in 293T cells in presence of wt or Y224A Rep. Varying volumes of crude lysate (in µl, x-axis) were added to HeLa cells and the percentage GFP positive -infected- cells was determined by FACS analysis. Data from four experiments are represented as mean ± s.e.m.

Mentions: To assess if the disruption of oligomerization observed with the Rep68Y224A mutant has any consequences on the AAV viral life cycle, we produced recombinant AAV2 particles expressing the GFP gene in presence of a helper virus containing the Y224A mutation in the Rep ORF. The cells were harvested and lysed, and the crude lysate (treated with an endonuclease) was used to infect Hela cells. Strikingly, the crude lysate from cells transfected with the mutant helper plasmid didn't contain any infectious rAAV2-GFP particles, as determined by FACS analysis of GFP positive cells (Figure 9). These results show that the residue Y224 of AAV Rep proteins, and the oligomeric properties it confers to these proteins, have a crucial role during the AAV life cycle.


The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

Zarate-Perez F, Bardelli M, Burgner JW, Villamil-Jarauta M, Das K, Kekilli D, Mansilla-Soto J, Linden RM, Escalante CR - PLoS Pathog. (2012)

Effect of Y224 mutation on AAV-2 virus liability.Comparison of the production of rAAV2-GFP infectious particles in presence of wt (squares) or Y224A Rep (triangles). rAAV2-GFP particles were produced in 293T cells in presence of wt or Y224A Rep. Varying volumes of crude lysate (in µl, x-axis) were added to HeLa cells and the percentage GFP positive -infected- cells was determined by FACS analysis. Data from four experiments are represented as mean ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375335&req=5

ppat-1002764-g009: Effect of Y224 mutation on AAV-2 virus liability.Comparison of the production of rAAV2-GFP infectious particles in presence of wt (squares) or Y224A Rep (triangles). rAAV2-GFP particles were produced in 293T cells in presence of wt or Y224A Rep. Varying volumes of crude lysate (in µl, x-axis) were added to HeLa cells and the percentage GFP positive -infected- cells was determined by FACS analysis. Data from four experiments are represented as mean ± s.e.m.
Mentions: To assess if the disruption of oligomerization observed with the Rep68Y224A mutant has any consequences on the AAV viral life cycle, we produced recombinant AAV2 particles expressing the GFP gene in presence of a helper virus containing the Y224A mutation in the Rep ORF. The cells were harvested and lysed, and the crude lysate (treated with an endonuclease) was used to infect Hela cells. Strikingly, the crude lysate from cells transfected with the mutant helper plasmid didn't contain any infectious rAAV2-GFP particles, as determined by FACS analysis of GFP positive cells (Figure 9). These results show that the residue Y224 of AAV Rep proteins, and the oligomeric properties it confers to these proteins, have a crucial role during the AAV life cycle.

Bottom Line: We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers.We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases.Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America.

ABSTRACT
The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

Show MeSH
Related in: MedlinePlus