Adenovirus

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Adenovirus

Adenovirus is an icosahedral non-enveloped (possessing no phospholipid membrane/envelope) DNA virus about 60-90 nm in diameter. The capsid is made of 252 capsomeres (240 hexons making up the faces and 12 pentons occupying the vertices). There is a spike at each penton vertex. The genome consists of linear dsDNA (double-stranded DNA) with bound basic proteins which condense the DNA for packaging into the capsid (the basic proteins neutralise the acidic charges normally found on DNA, reducing electrostatic repulsion between different regions of the DNA molecule). The genome is 35-36 kbp long (depending on adenovirus type) with inverted terminal repeats (ITRs) about 100 bp long at each end.

In humans, adenovirus causes primarily infections of the upper respiratory tract (including 5-10% of such infections in children) including common colds (although they are not the major cause of these) and bronchitis. They may also infect the lower respiratory tract, causing pneumonia (again not the major cause). They are also responsible for some cases of conjunctivitis, cystitis and gastroenteritis ('tummy' upsets). There are 52 serotypes (strains) that infect humans. Serotype 14 is potentially lethal and

adenovirus-36 has been linked to obesity, both statistically in humans (meaning it is more common in obese people) and as an infectious cause of obesity in various animal models (it induces a fat-gain syndrome in various animals, and transfusion of blood from an infected animal passes this infection on to another, an

example of Koch's postulates in action).

Koch's Postulates

To determine the causal agent of an infectious disease, experiments must satisfy Koch's postulates:

1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy animals.

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and

identified as being identical to the original specific causative agent.

Other adenoviruses infect other animals, e.g. Mastadenovirus infects mammals, Aviadenovirus infects birds, Atadenovirus infects mammals, birds and reptiles and Siadenovirus birds and frogs.

Adsorption and Entry

As usual, the first step in the infection-cycle is gaining access to the host cell. The 12 spikes of the

adenovirus capsid are adhesion receptors which recognise and bind to specific glycoprotein receptors on the target cell membrane (rather like an enzyme recognising its substrate). These bind to a glycoprotein on the target cell membrane called CAR (cysteine-aspartic protease or cysteine-dependent aspartate-directed protease). This initially adhesion is temporary and insufficient, but integrins on the target cell surface recognise and bind irreversibly to the penton at the base of the spike. Integrins are glycoproteins involved in cell adhesion and cell signalling. In this case, binding of adenovirus to the integrin causes the virus to be taken-up by the cell (which is fooled into treating the virus as a normal integrin ligand) by coated-pit (a type of receptor-mediated) endocytosis. The cell-surface membrane invaginates, forming a pit, which is coated on the cytoplasmic side by molecules of a cell-protein called clathrin. This pit invaginates and pinches off as

a vesicle in the cytoplasm - a ball of membrane, coated by clathrin and containing the virus.

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Uncoating

Cells normally process the contents of coated-vesicles in several different ways. One such way, and the one used in this case, is to send the vesicle to an endosome. Primary endosomes are bunches of vesicles and connecting tubules. Once the vesicle joins this mass it undergoes processing and the endosome matures into a secondary endosome. Secondary endosomes normally join with vacuoles called lysosomes and the resultant endo-lysosome functions as the cell's 'stomach' - acid and digestive enzymes are

released into it. Adenovirus, however, is not destroyed by this. Instead, when the endosome becomes more acidic (its pH drops) this triggers uncoating of the virus - the outer capsid dissassembles, revealing the viral DNA-protein core. The shed spikes have a toxic function and breach the membrane of the endosome, allowing the viral core to escape from the endosome into the cytosol of the host cell.

Delivery of DNA to the Cell Nucleus

Many viruses would remain in the cytosol and complete their cycle there, but adenovirus has a different strategy - it delivers its DNA to the host cell nucleus. It uses the cell's internal 'monorail' transport system of microtubules to carry it to the nucleus, where it arrives at and interacts with a nuclear pore complex (NPC). The NPC is a complex protein machine or gate that controls the entry and exit of materials to and from the nucleus. The virus triggers this gate to open and its naked DNA enters the nucleus. Once inside the nucleus, the DNA associates with the host's histone proteins, behaving like host DNA, and then transcription and synthesis of early proteins begins. These proteins inactivate host defenses and

synthesise viral DNA. The first viral protein produced, E1A activates other adenovirus promoters, resulting in the transcription of the early genes (E1B, E2A, E2B, E3 and E4.

DNA replication is illustrated below. The original genome has a protein, called TP (terminal protein) bound to the 5' end of each strand of the DNA duplex. A dC is bound to each TP. Adenovirus

polymerase binds and reads the template strand (in the direction 3' to 5') synthesising a new strand in the 5' to 3' direction, which displaces the old 5' to 3' strand. DNA polymerases require a short segment of dsDNA to initiate synthesis, which poses a potential problem here, however the TP-dC bound to the 5'

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strand mimics dsDNA, allowing the polymerase to begin synthesis. The polymerase binds along with

pTP-dC, pTP is preterminal protein, which is later converted into TP by adenovirus protease (this occurs late in the infection cycle and means that late DNA replication may involve DNA with pTP-dC bound at each end, rather than TP-dC; hence the diagram below is for early replication).

The displaced 5'-3' strand then forms a 'pan-handle' duplex - the genome has inverted terminal repeats that are complementary to one-another (depicted here by ABC and ZYX) and this creates a

short dsDNA region (duplex) for initiation of synthesis - adenovirus polymerase can bind this duplex, read the strand from 3' to 5' and hence synthesise the complementary 5'-3' strand. Thus we begin with one linear dsDNA (A in the diagram above) and finish each replication cycle with two (E and H). Recall that in prokaryotes and eukaryotes, DNA synthesise is bidirectional, beginning at an ori (origen of replication) somewhere in the middle of the DNA duplex and proceeding in both directions. In contrast, adenovirus

DNA synthesis is unidirectional and occurs at one or other end of the molecule (there is an ori at each end) and hence requires the TP mechanism to initiate polymerase action.

Early Gene Expression

As explained above, the first gene to be transcribed is the E1A gene which produces the E1A protein, which activates the transcription of the other early genes: The first viral protein produced, E1A activates other adenovirus promoters, resulting in the transcription of the early genes, E1B, E2A, E2B, E3 and E4. Some of the main functions of these early gene products are summarised below:

E1B. Two proteins are produced from the E1B gene, and both work together to prevent cell lysis during virus replication (specifically it inactivates the host cell protein p53 which initiates

programmed cell death, such as when the cell is damaged or infected!). E1B is also implicated in host cell transformation (that is it transforms the host cell into a tumour cell).

E2A is a single-stranded DNA binding protein (DBP) involved in DNA replication and transcription (presumably it stabilises single-stranded DNA when the duplex unzips; it requires zinc and so may have zinc-fingers with which to grab hold of the DNA molecule).

E2B is the adenovirus DNA polymerase, required for DNA synthesis. It also produces the pTP (precursor for the terminal protein).

E3 blocks the signal that virus-infected cells normally advertise on their cell-surface membranes (the major histocompatability complex, MHC I). If a passing natural killer (NK) cell, which is a

specialised cell in the immune system, detects such a signal then it destroys the virus-infected cell, preventing the virus from completing its replication-cycle. Clearly it benefits the virus to prevent this destruction!

E4 may be involved in oncogenesis (see below).

Note that some adenovirus genes can each produce a number of different proteins! Again this illustrates the economy of genetic information in viruses, in which DNA must be kept small to allow it to be packaged into a small capsid for efficient virus replication (viruses tend to maximise their number of progeny to increase their odds of finding and infecting new cells and so economisation on proteins and nucleotides has been extreme in virus evolution). This is achieved by splicing the primary transcript. The primary transcript is the RNA molecule initially produced by gene transcription. This transcript is modified to

produce mRNA (messenger RNA) for translation by the host ribosomes. Splicing of RNA is a process in which various regions of the RNA molecule can be removed, producing a new and smaller transcript. For example, splicing of the E4 primary transcript is thought to produce some six different

polypeptides/proteins (and all from one gene!). The part of a gene that actually encodes a protein (or polypeptide) is called an orf (open reading frame) and these adenovirus genes each have several orfs.Oncogenesis

Some adenovirus serotypes are capable of inducing tumour growth. This involves the genes E1A, E1B and sometimes E4. E1A causes uncontrolled DNA synthesis and cell division. As explained, E1B prevents programmed-cell death, which is necessary as when E1A triggers uncontrolled DNA synthesis, the cells would normally counter this loss of control by self-destructing, but E1B prevents this. Presumably the induction of tumour growth benefits the virus, perhaps by putting the cells into the necessary state, or by providing new cells containing the virus in a dormant state which can then be shed later, increasing the numbers of viral progeny for shedding from the host animal and infection of new animals. in any case, the tumour must be a suitable home for the virus. As research into tumour and cancers continues, it is

becoming apparent that many of these cancers are triggered by microbial infection, and virus infection in particular. A similar phenomenon occurs in plants. For example, the well known crown galls on the trunks of trees are tumours induced by the infective bacterium Agrobacterium tumefaciens in which the proliferating tissue of the gall provides shelter and nourishment to the bacterial parasite.

Late Gene Expression

Late in infection, the priority for the virus is not genome replication, but production of virus particles