AAV Structure: Detailed Look at Capsid Assembly

Adeno-associated viruses (AAVs) play a crucial role in gene therapy, serving as an effective vehicle for delivering genetic material due to their non-pathogenic nature and ability to mediate long-term expression. Understanding AAV capsid assembly is vital for optimizing vector efficiency and specificity.

Capsid Organization

The AAV capsid, with its icosahedral symmetry, consists of 60 protein subunits, an arrangement that is both structurally and functionally significant. This precise organization allows the virus to encapsulate its genetic material and interact with host cells. Each subunit contributes to capsid integrity, ensuring it can withstand external environments and deliver its contents efficiently. The arrangement of structural proteins follows a pattern revealed through cryo-electron microscopy, highlighting regions critical for receptor binding and immune evasion. The capsid’s surface features facilitate interaction with host cell receptors, aiding in attachment and internalization. This structural sophistication enables conformational changes essential for genome release inside the host cell.

Structural Proteins

The structural proteins of the AAV capsid are essential for its assembly and function, forming the capsid’s icosahedral structure and facilitating host cell interaction.

VP1

VP1, the largest AAV capsid protein, is crucial for viral infection. Its phospholipase A2 (PLA2) domain helps the virus escape from the endosome, a key infection step. Mutations in this domain can impair viral infectivity. VP1, comprising about 10% of capsid proteins, is strategically positioned for successful genome delivery.

VP2

VP2 acts as a structural bridge within the capsid, linking VP1 and VP3. It constitutes 10-15% of the capsid proteins and contributes to capsid stability and assembly. While lacking enzymatic activity, VP2’s presence is crucial for proper capsid formation, preventing incomplete or malformed capsids.

VP3

VP3, the most abundant capsid protein, forms the structural backbone, providing the framework for viral particle assembly. Comprising approximately 80% of the capsid proteins, VP3 is vital for capsid stability and genome packaging. Its structure has been extensively studied, revealing its role in forming the capsid’s outer shell.

Assembly Steps

AAV capsid assembly is a meticulously orchestrated process beginning with the synthesis of structural proteins VP1, VP2, and VP3. The specific ratio of these proteins ensures proper capsid assembly. As proteins are synthesized, they self-assemble into a pre-capsid structure, driven by their inherent properties and assisted by cellular chaperones. This process ensures capsid formation with the required icosahedral symmetry. The pre-capsid undergoes maturation, solidifying its structure and making it more resistant to environmental stresses. Post-translational modifications of capsid proteins, such as phosphorylation and acetylation, influence this maturation, affecting capsid stability and genome encapsulation.

Genome Encapsulation

AAV genome encapsulation is a finely-tuned process ensuring the virus functions as an effective gene delivery vector. The single-stranded DNA genome, synthesized in the host cell’s nucleus, is equipped with inverted terminal repeats (ITRs), crucial for recognition and packaging. AAV Rep proteins mediate packaging by recognizing ITRs and facilitating genome insertion into the pre-assembled capsid. This specific interaction ensures correct genetic material encapsulation, with Rep proteins unwinding DNA for efficient packing.

Serotype Variations

AAV serotype diversity is key to their gene therapy utility, with each serotype offering unique properties for specific therapeutic applications. Serotypes differ in capsid proteins, affecting their cell type tropism and immune response. Understanding these variations allows researchers to tailor AAV vectors for targeted tissue delivery, enhancing gene therapy precision. AAV2, for example, has broad tropism due to its affinity for heparan sulfate proteoglycans, but its widespread nature can trigger immune responses. Other serotypes, like AAV8 and AAV9, offer reduced immunogenicity and enhanced ability to cross the blood-brain barrier, advantageous for neurological disorder treatment. Engineered capsids, developed through directed evolution and rational design, offer customized properties, such as enhanced stability or improved transduction efficiency, addressing challenges like pre-existing immunity. This customization potential advances gene therapy, providing vectors tailored to diverse patient needs.