CONTENTS:

Molecular Biology

Summary

Viral Replication

Adenovirus replication can be divided into two phases: early events and late events. The transition between these two phases occurs when viral genome replication commences. The early phase of viral infection lasts five to six hours while the late phase can last up to 24 hours. A single virus can produce upwards of 10,000 progeny.

The first step in any successful viral infection is adsorption. Attachment to the host cell is mediated by the carboxy-terminus of the fiber protein (the terminal knob). It is thought that different serotypes of this virus group may bind different receptors, as fiber length is dependent on serotype. Two receptors known to associate with the fiber protein are the MHC class I molecule and the coxsackievirus-adenovirus receptor. In addition to the fiber, a second protein interaction must occur to promote internalization of the virus. The base of the penton interacts with cell surface integrins. Once this interaction occurs, the virus may be internalized by receptor mediated endocytosis. The virus is then encased in an endosome within the cytosol. Acidification of the endosome allows the virus to escape into the cytosol. It is thought that the penton base somehow promotes this acidification.

The virus then utilizes the cellular microtubule network to reach the nucleus. By the time the virus reaches the nucleus, it has shed its capsid in an organized manner. The pentons with their associated hexons are removed before the remaining hexons and their associated proteins. The terminal protein helps associate the viral DNA to the nuclear matrix and in some way this helps initiate transcription.

As the adenovirus enters the early stages of gene expression, it must accomplish three things. The virus must induce the host cell to enter the S phase of the cell cycle, establish viral systems that protect against host defenses and synthesize products needed for viral DNA replication.

The E1A gene is the first gene to be transcribed in the nucleus. Host cell RNA polymerase II is used to carry out this early transcription. This gene encodes two mRNA’s for early events. These two transcripts differ by a 46 amino acid sequence due to differences in splicing and are called the 12S and 13S proteins. By binding to regulatory and transcription factors, these two proteins initiate transcription of the other transcriptional units. In combination with E2F, these proteins are responsible for inducing the cell to remain in S phase, which is favorable for viral replication. Once transcribed, E2F in combination with E1A will bind the cellular retinoblastoma tumor suppressor protein (pRB). The E1B protein will bind the p53 gene to alter cell cycle progression. The pRB-E2F complex and the E1B-p53 complex help regulate the cell cycle such that it enters and remains in S phase. Essentially, these proteins disrupt elements that normally regulate the cell cycle in order to make conditions most favorable to viral replication.

Translation of the early mRNA’s is carried out on host polysomes in the cytoplasm. At this stage, there is no competition between viral mRNA and host mRNA. However, by the time the viral infection has progressed to the late stage, viral mRNA will outcompete host mRNA at these complexes.

The virus is ready to begin replication (late events) once the cell enters the S phase and adequate products from the E2 gene have accumulated. Replication occurs in two stages and starts at one of the origins located within a terminal repeat. One strand of the genome will serve as a template and replication will occur down the entire length of the DNA. At least three proteins encoded by the virus are known to have a role in replication. No primer is needed as the covalently bound terminal protein (TP) is used for this purpose. The virus also provides Ad DBP that is a DNA-binding protein and Ad DNA Pol that is a DNA-dependent polymerase for its replication. Cellular proteins are also used during genome replication.

The initial stage of replication results in a double stranded DNA genome and a displaced single strand. The second stage of replication involves synthesis along this displaced strand. Due to the inverted repeats at the termini, the DNA is able to circularize forming a panhandle structure. The machinery for replication recognizes this panhandle to be identical to the initial site of replication. Synthesis of the complementary strand provides a complete genome copy.

Sometimes virus replication takes a different pathway (minor pathway). In this pathway, replication is initiated at both origins of replication. Once the middle of the molecule is reached, the unit will fall apart into two replicative intermediates. The machinery for replication recognizes the panhandle structure and will complete replication.

The part of the adenovirus genome coding for late event proteins is a single large transcript. Various splicing of this region produces 18 mRNA’s that can be categorized into five families (L1 to L5). These proteins are translated in the cytoplasm and then transported back into the nucleus. Capsid proteins accumulate in the nucleus and assemble. The DNA contains a packaging sequence that permits recognition between the capsid and the DNA. One or more unidentified proteins are thought to aid in this recognition event. As no empty capsids will accumulate within the cell, capsid assembly and DNA packaging is thought to be a simultaneous event. The exact mechanism by which this occurs remains a subject of controversy.

Once assembled, the virus must disrupt the intermediate filament system to escape the host cell. This system help maintain the mechanical integrity of the cell. Once disrupted, the cell may be lysed and the virus may escape.