Adenovirus
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.
Adenovirus uncoating
Adenovirus adsorption
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'
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
(virions). Capsids must be produced and the viral DNA packaged into them, ready for release from the
host cell. The late viral proteins, that is those produced late in infection, are structural proteins and
proteins involved in binding to and penetration of new host cells. These are:

  • Protein II which produces hexons
  • protein III, the penton base and IIIa which is associated with the penton base
  • IV, the fiber or spike involved in receptor binding
  • V part of the core, associates with the viral DNA and the penton-base (a histone-like protein)
  • VI a minor component of the hexon which may be involved in capsid assembly, maturation or
    stabilisation
  • VII part of the core and histone-like
  • VIII another minor component of the hexon, may have similar functions to VI
  • IX another minor component of the hexon, may have similar functions to VIII and VI

Also, packaging involves the genome-binding TP (terminal protein) derived from the pTP produced by
E2B.

Assembly and Packaging

The core consists of one molecule of TP covalently bonded to each 5' end of the genomic DNA molecule;
180 copies of protein V and 1070 of VII and a small protein called Mu, of unknown function and location.
Small basic proteins (possibly mu) neutralise the negative charges of the DNA, allowing the DNA to be
more tightly compacted (like charges repel!).

Escape from the Host Cell

The viral protein E3 is the adenovirus death protein. This accumulates during the course of infection
of the cell and at a very late stage induces cell lysis by switching-on the cell's own self-destruct
mechanism: the cell undergoes
autophagy (literally 'eats' itself as its lysosomes digest cell organelles)
and the cell protein
caspase (cysteine-aspartic protease or cysteine-dependent aspartate-directed
protease) is activated. Caspase initiates
apoptosis (programmed cell death). At this very late stage, new
virion particles have accumulated in the nucleus, but as the cell self-destructs it lyses (bursts open)  and
the virus progeny are freed to infect other cells and begin the cycle again.

Conclusion

The above account is a summary only of some key aspects of adenovirus biology. It serves to illustrate
how complicated viruses can be, despite their having so few genes and components. The clever tricks
that viruses employ to economise on genetic material, to take control of host cells and evade the host's
immune system do cause immense grief, but are nevertheless beautiful examples of the prowess of
Nature as an information technologist, mechanical, chemical and molecular engineer! It also illustrates
how extensive research, which is ongoing, is needed to understand even a single virus and its diseases,
but at the same time, we can see that certain common themes and strategies emerge, which are deployed
by many different viruses. Virology is truly a fascinating subject!


References

K.N. Leppard, 1997. E4 gene function in adenovirus, adenovirus vector and adenoassociated virus
infections.
Journal of General Virology 78: 2131–2138.

Brian P. McSharry, Hans-Gerhard Burgert, Douglas P. Owen, Richard J. Stanton, Virginie Prod'homme,
Martina Sester, Katja Koebernick, Veronika Groh, Thomas Spies, Steven Cox, Ann-Margaret Little, Eddie
C. Y. Wang, Peter Tomasec, and Gavin W. G. Wilkinson, 2008. Adenovirus E3/19K Promotes Evasion of
NK Cell Recognition by Intracellular Sequestration of the NKG2D Ligands Major Histocompatibility
Complex Class I Chain-Related Proteins A and B.
J. Virol. 82: 4585-4594

Sara Salinas, Lynsey G. Bilsland, Daniel Henaff, Anne E. Weston, Anne Keriel, Giampietro
Schiavo, Eric J. Kremer, 2009. CAR-Associated Vesicular Transport of an Adenovirus in
Motor Neuron Axons. PLoS Pathog 5: e1000442.
animated adenovirus
adenovirus replication