Viral Replication

The Ad viral life cycle is understood best for subgroup C, which is another factor in the choice of Ad5 and 2 as gene therapy vectors (Fig. 3). The knob of the fiber protein binds to the CAR receptor (13), followed by an interaction of the RGD sequence in the penton base with cell surface aVp3 or aVp5 integrins (15). Excess soluble integrins inhibit Ad internalization but not binding, suggesting that penton base-integrin interaction is instrumental in internalization (16). Ad modified with deletion of the RGD motif replicate effectively in vitro so the penton base-integrin interaction is likely related to efficiency of Ad infection, but is not essential (17). The Ad enters the cell by endocytosis into clathrin-coated pits, a process that can be blocked by dy-namin inhibitors (18). After endocytosis, Ad is very rapidly released into the cytoplasm prior to extensive endosome fusion. The virus proceeds rapidly to the nucleus, probably actively transported on microtubules using the dynein motor (19), and then binds to the surface of the nucleus near the nuclear pore (20). Using fluorescent viruses, this process has been shown to be efficient and rapid, with >90% of Ad5 delivered to the nucleus within 1 hr (21). At the nuclear membrane, the DNA and terminal protein are internalized by an unknown mechanism, and are assembled into the nuclear scaffold for active transcription.

With wild-type Ad, the viral E1A gene is transcribed immediately after infection (10). After alternate splicing, the E1 mRNAs are translated into the two E1A proteins

Figure 2 Structure and transcription of the major genes of the adenovirus type 5 genome. Schematic summary of the transcription of adenovirus during lytic infection. The genome is represented as 2 parallel lines and is divided by the scale shown on top into 100 map units (1 map unit = 360 bp). There are 9 major complex transcription units divided into early (above the genome) and late transcripts (below). The 4 early transcripts are produced before the commencement of DNA replication and specify regulatory proteins and proteins required for DNA replication. Upon initial infection of a cell, the E1A protein is produced from transcripts in the E1 region. E1A is a major regulatory factor required for transcription of E1B, E2, E3, and E4. In replication-deficient adenovirus vectors, the E1 region is deleted. Proteins coded by the E2 and E4 regions are required for late gene transcription. The E3 region codes for proteins that help the virus to evade host defenses. All late transcripts rightward originate at the same point and are produced by alternate splicing. The tripartite leader sequence is present at the 5' end of all late transcripts. The L3 region specifies hexon, the L5 region specifies fiber, and the L2 region specifies penton.

Figure 2 Structure and transcription of the major genes of the adenovirus type 5 genome. Schematic summary of the transcription of adenovirus during lytic infection. The genome is represented as 2 parallel lines and is divided by the scale shown on top into 100 map units (1 map unit = 360 bp). There are 9 major complex transcription units divided into early (above the genome) and late transcripts (below). The 4 early transcripts are produced before the commencement of DNA replication and specify regulatory proteins and proteins required for DNA replication. Upon initial infection of a cell, the E1A protein is produced from transcripts in the E1 region. E1A is a major regulatory factor required for transcription of E1B, E2, E3, and E4. In replication-deficient adenovirus vectors, the E1 region is deleted. Proteins coded by the E2 and E4 regions are required for late gene transcription. The E3 region codes for proteins that help the virus to evade host defenses. All late transcripts rightward originate at the same point and are produced by alternate splicing. The tripartite leader sequence is present at the 5' end of all late transcripts. The L3 region specifies hexon, the L5 region specifies fiber, and the L2 region specifies penton.

Figure 3 Trafficking of adenovirus from membrane to nucleus. The initial contact between the virus and cell is mediated by the knob of fiber and the coxsackie adenovirus receptor (CAR). This allows the secondary interaction between the penton and aVp3 or aVp5 integrins, which is required for internali-zation. The initial internalization is via coated pits, which give rise to coated vesicles. After a very short interval, prior to fusion of early endosomes into sorting endosomes, a conforma-tional change in the viral capsid allows escape of the virus into the cytoplasm. Microtubules carry the virus toward the nucleus. The whole capsid attaches to the outside of the nucleus but only the DNA and terminal protein are inserted into the nucleus itself, where they are assembled onto the nuclear matrix to allow transcription. (Courtesy of P. Leopold, Weill Medical College of Cornell University, New York, NY.)

Figure 3 Trafficking of adenovirus from membrane to nucleus. The initial contact between the virus and cell is mediated by the knob of fiber and the coxsackie adenovirus receptor (CAR). This allows the secondary interaction between the penton and aVp3 or aVp5 integrins, which is required for internali-zation. The initial internalization is via coated pits, which give rise to coated vesicles. After a very short interval, prior to fusion of early endosomes into sorting endosomes, a conforma-tional change in the viral capsid allows escape of the virus into the cytoplasm. Microtubules carry the virus toward the nucleus. The whole capsid attaches to the outside of the nucleus but only the DNA and terminal protein are inserted into the nucleus itself, where they are assembled onto the nuclear matrix to allow transcription. (Courtesy of P. Leopold, Weill Medical College of Cornell University, New York, NY.)

essential for transcription of other early viral mRNAs. E1A proteins promote the expression of cellular genes needed for DNA replication by interacting with the retinoblastoma susceptibility protein (Rb), which normally suppresses entry into S phase of the cell cycle by complexing with the host transcription factor E2F. E1A also interacts with numerous cellular transcriptional factors to promote the assembly of complexes that promote transcription of other early adenovi-ral genes. Among the important downstream products induced by E1A is the product of the E1B gene, which blocks the apoptotic pathway through interaction with p53 long enough for a productive viral infection. The E1B 55-kDa protein also complexes with the ORF6 protein from the E4 region to modulate expression of the viral late genes, which begin to be expressed around 6 hr postinfection. At that time, DNA replication begins and the transcription of late genes commences, providing the capsid components that assemble into mature virions (10). The new virions are assembled in the nucleus, necessitating transport of capsid proteins into the nucleus. As the viral infection proceeds, the integrity and viability of the cells decreases, but the mechanism of viral release from the cell is not understood.

In the context of gene therapy, the E3 region is important as it encodes immunosuppressive functions that work through two mechanisms (22). The E3 gp19-kDa protein prevents major histocompatibility complex (MHC) class I-mediated antigen presentation on the cell surface, thereby inhibiting the differentiation of cytotoxic T lymphocytes directed against viral antigens (22). The E3 14.7-kDa and E3 10.4-kDa proteins inhibit apoptosis of infected cells initiated by fas/fas ligand and/or tumor necrosis factor (23). The promoter for the E3 region requires E1 products, and thus in E1" deleted Ad vectors, the presence or absence of the E3 region is not relevant (see below).

Transcripts from the E2 region specify the three nonhost proteins directly involved in DNA replication: the DNA polymerase, the single-stranded DNA-binding protein (ssDBP) and the preterminal protein (10). Like other viruses, adenovirus has developed a specific strategy for the faithful replication of the ends of its DNA. The last 103 nt at both ends of the genome consist of inverted copies of the identical sequence. The terminal protein binds covalently to the 5' end and acts as a primer for DNA synthesis by the adenoviral DNA polymerase of the leading strand starting at either end. DNA polymerase proceeds by a strand displacement mechanism, creating a duplex and a displaced strand that is sequestered by the ssDBP and has terminal protein attached to one end. Base pairing of the ends of the single strand creates a panhandle structure with ends identical to those of the duplex. Reformation of duplex from the single-stranded form occurs by the same mechanism with the Ad polymerase initiating at the terminal protein and displacing the ssDBP. Interestingly, the viral genomes undergoing replication are at a different location from those being transcribed (24). DNA is packaged into capsids as directed by a DNA sequence close to the left-hand end of the virus. The efficiency of packaging depends on the length of DNA; genomes greater than 105% or less than 95% of the normal length propagate much less efficiently (25,26).

The E4 region plays important roles in the viral life cycle by promoting the selective expression of viral genes at the expense of cellular genes. For example, the E4-ORF3 and ORF6 proteins inhibit the transport of transcripts of cellular genes from nucleus to cytoplasm while promoting the transport of late viral transcripts (10). The E4 region is therefore essential for viral gene expression and subsequent viral replication.

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