AAV Structure and Function: Detailed Insights

Adeno-associated viruses (AAVs) have emerged as versatile tools in gene therapy, offering solutions to numerous genetic disorders. Their appeal largely stems from their relatively low immunogenicity and the ability to target specific tissues effectively.

Given the increasing reliance on AAVs within both clinical and research settings, it becomes crucial to delve into their structural intricacies and functional mechanisms.

Capsid Proteins

The capsid proteins of adeno-associated viruses (AAVs) are fundamental to their structure and function, playing a pivotal role in their ability to deliver genetic material to target cells. Composed of 60 protein subunits, the AAV capsid is primarily made up of three viral proteins: VP1, VP2, and VP3. These proteins are encoded by the cap gene and are expressed in a specific ratio, with VP3 being the most abundant. The unique assembly of these proteins forms an icosahedral structure, which is critical for the virus’s stability and infectivity.

Each of the capsid proteins contributes distinct functionalities. VP1, the largest of the three, contains a phospholipase A2 (PLA2) domain, which is essential for the virus to escape from the endosome after entry into the host cell. This domain is hidden within the capsid until the virus is inside the cell, ensuring that the PLA2 activity is only triggered at the appropriate time. VP2, though less understood, is believed to play a role in the proper assembly of the capsid and may assist in the incorporation of VP1 into the capsid structure. VP3, the smallest and most numerous, forms the bulk of the capsid and is responsible for the overall architecture and stability of the virus.

The surface topology of the AAV capsid is decorated with protrusions and depressions that are crucial for its interaction with host cell receptors. These surface features determine the tropism of the virus, meaning they influence which cell types the virus can infect. For instance, specific amino acid residues on the capsid surface can bind to different cellular receptors, such as heparan sulfate proteoglycans or sialic acid, guiding the virus to its target cells. This receptor binding is the first step in the virus’s entry process, making the capsid proteins indispensable for successful infection.

Genome Organization

The genome of adeno-associated viruses (AAVs) is a single-stranded DNA (ssDNA) molecule, approximately 4.7 kilobases in length. This compact genome is flanked by two inverted terminal repeats (ITRs), which are crucial for the replication and packaging of the viral DNA. The ITRs form T-shaped hairpin structures that serve as origins of replication and are recognized by the viral replication machinery.

Within the AAV genome, there are two main open reading frames (ORFs): rep and cap. The rep gene encodes the Rep proteins, which are involved in viral replication, transcriptional regulation, and packaging of the viral genome. These proteins are multifunctional, with Rep78 and Rep68 being responsible for initiating replication and Rep52 and Rep40 playing a role in unwinding the DNA during replication. The precise regulation of these proteins ensures the efficient production of viral particles.

The cap gene, on the other hand, is responsible for encoding the capsid proteins that form the virus’s outer shell. The expression of the cap gene is tightly regulated by alternative splicing and different promoters, allowing the virus to produce the three capsid proteins in the required stoichiometric ratios. This regulation is essential for the proper assembly of the viral capsid and, consequently, the infectivity of the virus.

Interestingly, AAVs have a unique dependency on helper viruses, such as adenoviruses or herpesviruses, for their replication. In the absence of a helper virus, AAVs can establish a latent infection by integrating their genome into the host cell’s DNA, specifically at a site in chromosome 19 called AAVS1. This integration is mediated by the Rep proteins and allows the virus to persist in a quiescent state until helper virus co-infection reactivates replication.

Serotypes and Variants

Adeno-associated viruses (AAVs) exhibit considerable diversity, with multiple serotypes and variants that enable them to target a wide range of tissues and cells. This diversity is fundamentally shaped by the genetic variations in their capsid proteins, which influence their ability to bind to different cellular receptors. Serotypes are classified based on their immunological properties and tissue tropism, with AAV1 through AAV9 being the most studied. Each serotype has unique characteristics that make it suitable for specific therapeutic applications.

For instance, AAV2 is one of the most extensively researched serotypes and is known for its ability to transduce a variety of cell types, making it a versatile tool in gene therapy. AAV8, on the other hand, has shown a strong affinity for liver cells, making it particularly useful for treating liver-related genetic disorders. This tissue-specific targeting is achieved through the unique interactions between the capsid proteins and cellular receptors, highlighting the importance of selecting the appropriate serotype for a given therapeutic goal.

The development of AAV variants has further expanded the potential of these vectors. Through techniques such as directed evolution and rational design, researchers have engineered novel AAV variants that exhibit enhanced properties, such as increased transduction efficiency, reduced immunogenicity, and the ability to evade pre-existing antibodies. Directed evolution involves creating a library of capsid mutants and selecting those with the desired traits, while rational design uses structural knowledge to make specific modifications to the capsid proteins. These advancements have led to the creation of next-generation AAV vectors that are more efficient and safer for clinical use.

In addition to naturally occurring serotypes and engineered variants, cross-packaging strategies have also been employed to broaden the utility of AAV vectors. Cross-packaging involves encapsulating the genome of one serotype within the capsid of another, combining the advantageous properties of both. This strategy can enhance the transduction capabilities and tropism of the virus, providing greater flexibility in designing gene therapy vectors tailored to specific needs.

Host Cell Entry

When adeno-associated viruses (AAVs) initiate the process of host cell entry, they first encounter the extracellular matrix, where they bind to specific cell surface receptors. This initial binding is highly selective, dictated by the unique surface topology of each AAV variant, which determines its affinity for certain cell types. Once the virus attaches to its primary receptor, it often engages additional co-receptors that facilitate its internalization through endocytosis.

Following receptor-mediated endocytosis, AAVs are enveloped in endosomes, cellular vesicles that transport the virus deeper into the cell. Within these acidic compartments, conformational changes in the viral capsid are triggered, exposing domains essential for the next step in the entry process. Endosomal escape is a critical juncture, as it allows the viral genome to avoid degradation within lysosomes. The virus employs various strategies, including the use of endosomal membrane-disrupting peptides, to breach the endosomal barrier and release its genetic payload into the cytoplasm.

Once in the cytoplasm, the single-stranded DNA genome of AAV must be converted into double-stranded DNA to facilitate transcription and subsequent gene expression. This conversion is carried out by host cell machinery, which recognizes the viral genome and initiates replication. Successful conversion and transcription are prerequisites for the AAV to exert its therapeutic effects, as the encoded genes must be expressed at sufficient levels to achieve clinical efficacy.