Adeno-Associated Virus (AAV) is a small, non-enveloped virus that has garnered significant attention in the field of gene therapy. Its outer shell, known as the capsid, provides protection for its genetic material and plays a fundamental role in its interaction with host cells. This structure is assembled from three distinct viral proteins: VP1, VP2, and VP3. These proteins are essential for the virus’s ability to infect cells and deliver its genetic payload.
Understanding the AAV Capsid
The AAV capsid serves as an outer shell, safeguarding the viral genetic material, a single-stranded DNA molecule approximately 4.7 kilobases long. This protective shell is formed by the assembly of 60 individual protein subunits—VP1, VP2, and VP3—into an icosahedral structure with T=1 symmetry. The three proteins share a common C-terminus but differ in their N-termini, arising from a single cap gene through alternative splicing and different start codons.
The relative abundance of these proteins within the assembled capsid is a molar ratio of 1:1:10 for VP1, VP2, and VP3, respectively. A single AAV capsid contains about 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3. This stoichiometry is maintained by translation mechanisms. The arrangement of these proteins contributes to the capsid’s structural stability and integrity, necessary for its survival outside a host cell and for successful delivery of its genetic cargo.
Distinct Functions of VP1, VP2, and VP3
Each of the three capsid proteins, VP1, VP2, and VP3, contributes distinct functions to the AAV life cycle. While all three are part of the assembled virion, their distinct N-terminal regions dictate their specialized roles in cellular infection.
VP1, the largest of the three proteins, is important for the initial stages of infection after the virus enters a cell. Its N-terminus contains a phospholipase A2 (PLA2) domain and nuclear localization signals (NLSs). The PLA2 domain helps breach the endosomal membrane, allowing the virus to escape into the cytoplasm. The NLSs facilitate transport of the capsid into the host cell nucleus, where the genome is uncoated and expressed. These N-terminal regions are sequestered within the capsid but become exposed during infection, triggered by the acidic endosomal environment.
VP2’s role is less extensively characterized than VP1 or VP3, but it contributes to capsid assembly and stability. While not essential for particle formation, its presence can influence VP3’s nuclear localization. The N-terminus of VP2, like VP1, can externalize during infection, suggesting a role in intracellular trafficking or uncoating, though its mechanism is still under investigation.
VP3 is the most abundant structural protein of the AAV capsid. Its primary function is to provide structural integrity and stability to the viral particle. The common region shared by VP1, VP2, and VP3 is sufficient to assemble the virus capsid, highlighting VP3’s fundamental role in forming the basic scaffold. VP3 also plays a role in the initial stages of cell attachment and receptor binding, as the outer surface of the capsid, largely composed of VP3, interacts with host cell receptors like heparan sulfate proteoglycan for certain AAV serotypes.
Significance in Gene Therapy
The properties of AAV capsid proteins, including their low immunogenicity and ability to infect various cell types, make AAV an attractive vector for gene therapy applications. The capsid’s stability allows for efficient delivery of therapeutic genes to target cells. Researchers can modify these capsid proteins to overcome limitations like pre-existing immunity or to enhance targeting to specific tissues.
One approach involves rational design, where specific amino acid residues on the capsid surface are altered to change the virus’s tropism or reduce immune recognition. For example, adding positively charged residues like lysine to the capsid can enhance its ability to bind to negatively charged cell surface molecules, altering tissue targeting. Such modifications can de-target the virus from organs like the liver, where it naturally accumulates, and redirect it to other tissues, such as the lung.
Another strategy is directed evolution, which involves creating diverse AAV variants through random mutagenesis and selecting for desired properties. This method can lead to the discovery of capsids with improved transduction efficiency, enhanced ability to penetrate specific barriers like the blood-brain barrier, or reduced immunogenicity. By engineering VP1, VP2, and VP3 proteins, researchers optimize AAV vectors for safer, more effective gene delivery, addressing challenges like neutralizing antibodies and improving gene therapy success.