Structure

Adenovirus consists of an icosahedral protein capsid of approximately 70-100 nm diameter and, within that capsid, a single copy of a double-stranded DNA molecule of length approximately 36,000 bp [Fig. 1; reviewed in (10)]. In the context of gene therapy, the fiber, penton base, and hexon are the most important capsid proteins. The 20 triangular faces of the viral capsid are built from hexon, the major capsid protein. The 240 hexon capsomeres in the capsid are each trimers comprising 3 copies of the 105-kDa hexon subunit, with each trimer interacting with 6 others in a pseudoequivalent fashion. The 3-dimensional structure of hexon shows that the homotrimer has loops that project from the capsid surface (11). Capsid proteins VI, VIII, and IX are associated with hexon, and their role is to stabilize the capsid structure. The 12 capsid vertices are made up of the penton capsomere, a complex of 5 copies of the penton base, and 3 copies of fiber. Each penton capsomere interacts with 5 hexon capsomeres, 1 from each of the 5 faces that converge at the vertex. The fiber protein projects outward from the penton base. The DNA is wrapped in the histone-like core protein VII, and there is a terminal protein attached to the 5' end of each DNA strand.

Neutralizing antibodies are directed primarily against epi-topes located on the loops of the hexon. This is expected, as the loops project from the surface of the virus where they are accessible to antibodies. When the primary structures of the capsids of different serotypes are compared, related Ad differ most in these loops, suggesting the selective pressures applied by the immune system result in the emergence of mutations in the external hexon loops (12).

The fiber protein is a trimer consisting of 3 domains: the base, shaft, and knob. The N-terminal base domain interacts with the penton base. The shaft includes an ex tended domain consisting of variable numbers of a 15 amino acid pseudorepeat. The number of repeats, and therefore the length of the shaft, varies between 23 copies for the group A viruses and 6 copies for the subgroup B viruses. The distal C-terminal domain of the fiber protein, referred to as the ''knob,'' interacts with the high-affinity receptor on the surface of the target cell. The high-affinity receptor for adenoviruses except those of subgroup B is referred to as CAR (coxsackie-adenovirus receptor), reflecting the fact that the coxsackie B viruses and most serotypes of Ad share the same receptor (13,14). CAR is a single membrane-spanning protein with two extracellular immunoglobulin-like domains. Apart from acting as a virus receptor, the function of CAR is unknown.

A sequence motif on the penton base is involved in internalization of the virus after high-affinity CAR-fiber interaction. In serotypes 2 and 5 the amino acid motif arginine-glycine-aspartate (RGD) interacts with aVp3 and aVp5 integrins of the cell surface, and this interaction is essential for efficient internalization (15).

For adenovirus type 5, the most commonly used Ad for gene transfer vectors, the complete 35,935 bp DNA sequence is known. For convenient reference, the genome is divided into 100 equally sized map units. A detailed transcription map at various time points postinfection is used to divide the genome into interspersed early (E) and late (L) regions [Fig. 2; reviewed in (10)]. There is considerable transcrip-tional overlap among the genes, making manipulation of some areas of the genome difficult. Each of the 5 early genes is comprised of a complex transcription unit with alternative sites for transcription initiation, termination, and

Figure 1 Structure of the adenovirus capsid. Shown (left) is a 3-dimensional representation and (right) a simplified cross-section of the capsid showing the deployment of the capsid proteins and Ad genome. The capsid is an icosahedron with 20 faces and 12 vertices. The faces are composed of hexons, each comprised of trimers of the hexon protein. The hexons are trapezoid shaped, with three loops on top, extending from the face of the capsid. The loops represent the variable regions that differ among serotypes and are the major epitopes for neutralizing antibodies. Proteins IX and VIII are associated with the hexon and are believed to stabilize the capsid. The vertices are composed of a fiber and penton base. The fiber has 3 domains: the base that interacts with penton, the shaft, and the knob. The knob interacts with a high-affinity receptor on the target cell and the shaft holds the virus away from the surface of the cell, depending on the length of the shaft. The penton base interacts with the hexon and the fiber, and contains epitopes that interacts with integrins on the cell surface. The 36-kb double-stranded DNA genome is wrapped around capsid core protein VII and the terminal protein is attached to the two 5' ends of the Ad genome. (Adapted from Ref. 10.)

Figure 1 Structure of the adenovirus capsid. Shown (left) is a 3-dimensional representation and (right) a simplified cross-section of the capsid showing the deployment of the capsid proteins and Ad genome. The capsid is an icosahedron with 20 faces and 12 vertices. The faces are composed of hexons, each comprised of trimers of the hexon protein. The hexons are trapezoid shaped, with three loops on top, extending from the face of the capsid. The loops represent the variable regions that differ among serotypes and are the major epitopes for neutralizing antibodies. Proteins IX and VIII are associated with the hexon and are believed to stabilize the capsid. The vertices are composed of a fiber and penton base. The fiber has 3 domains: the base that interacts with penton, the shaft, and the knob. The knob interacts with a high-affinity receptor on the target cell and the shaft holds the virus away from the surface of the cell, depending on the length of the shaft. The penton base interacts with the hexon and the fiber, and contains epitopes that interacts with integrins on the cell surface. The 36-kb double-stranded DNA genome is wrapped around capsid core protein VII and the terminal protein is attached to the two 5' ends of the Ad genome. (Adapted from Ref. 10.)

splicing. The E1A and E1B genes are transcribed rightward at the left-hand end of the genome close to the DNA replication origin and DNA packaging signal. The E4 region is transcribed leftward at the right-hand end of the genome. Distal to the E1 and E4 regions are the termini of the DNA, which are inverted copies of the same sequence. Replication of the ends of the DNA is achieved by the attachment of terminal protein to the 5' end of the DNA, which acts as a primer to initiate unidirectional replication. This terminal protein is one of the components of the E2 transcriptional unit, which is transcribed leftward commencing at map unit 75. The remaining early transcription unit is the E3 gene, which is transcribed rightward commencing at map unit 77.

The 5 late genes are expressed after the beginning of DNA replication and encode the viral structural proteins. These late transcripts are all transcribed rightward originating from map unit 17 and contain the same 3-part leader sequence before alternate splicing generates different mature mRNAs.

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