Adenoviruses

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Introduction:

Adenoviruses are a frequent cause of acute upper respiratory tract (URT) infections, i.e. "colds". In addition, they also cause a number of other types of infection. They were first isolated in 1953 by investigators trying to establish cell-lines from adenoidal tissue of children removed during tonsillectomy and from military recruits with febrile illness.
In 1962, some Adenoviruses were shown to cause tumours in rodents - this caused a considerable panic! (N.B. Adenovirus oncogenesis appears to be associated with abortive infections and has never been observed in humans.)
During investigation of the Adenovirus genome and gene expression, many techniques were developed which were subsequently used to examine other viruses/cellular genes - these viruses are an important model system for the understanding of many other viruses. Some characteristic features of Adenoviruses are:

• Widespread in nature, infecting birds, many mammals and man. There are two genera, Aviadenovirus (avian) and Mastadenovirus (mammalian)
• Can undergo latent infection in lymphoid tissues, becoming reactivated some time later.
• Several types have oncogenic potential.

Taxonomy:

Morphology:

Detailed three dimensional structural models of adenovirus particles have been constructed based on a combination of cryoelectron microscopy and X-ray crystallography. There are at least 13 proteins in the Adenovirus capsid:

All Adenovirus particles are similar: non-enveloped, 60-90nm diameter. They have icosahedral symmetry easily visible in the electron microscope by negative staining and are composed of 252 capsomers: 240 "hexons" + 12 "pentons" at vertices of icosahedron (2-3-5 symmetry).
Individual protomers can be isolated by progressive chemical disruption of purified virus particles. The hexons consist of a trimer of polypeptide II with a central pore; VI, VIII and IX are minor polypeptides also associated with the hexon, thought to be involved in stabilization and/or assembly of the particle. The pentons are more complex; the base consists of a pentamer of peptide III, 5 molecules of IIIa are also associated with the penton base. The pentons have a toxin-like activity, purified pentons causing c.p.e. in the absence of any other virus components (a unique property). A trimeric fibre protein extends from each of the 12 vertices (attached to the penton base proteins) and is responsible for recognition and binding to the cellular receptor. A globular domain at the end of the adenovirus fiber is responsible for recognition of the cellular receptor.

To view a negatively-stained electron micrograph of adenovirus particles, click here. The thin fibres protruding from each vertex of the icosahedral particle are just visible (look closely!) and the triangular faces of the icosahedral particle can be made out. Image reconstruction of a type 2 adenovirus particle.

Genome:

Linear, non-segmented, d/s DNA, 30-38kbp (size varies from group to group) which has the theoretical capacity to encode 30-40 genes. Genome structure (cross-hybridization, restriction map) is one of the characters used to assign viruses to groups (70-95% homology within groups, 5-20% homology between groups).

• The terminal sequences of each strand are inverted repeats, hence the denatured single strands can form "panhandle" structures (100-140bp).
• There is a 55kD protein covalently attached to the 5' end of each strand.

Replication:

Replication is divided into EARLY and LATE phases, the latter defined as beginning with the onset of DNA replication (N.B. this division is characteristic of the replication of DNA viruses!). Attachment to cells is rather slow, taking several hours to reach a maximum.

Uptake of the adenovirus particle is a two stage process involving an initial interaction of the fibre protein with a range of cellular receptors, which include the MHC class I molecule and the coxsackievirus-adenovirus receptor. The penton base protein then binds to the integrin family of cell surface heterodimers allowing internalization via receptor-mediated endocytosis. Most cells express primary receptors for the adenovirus fibre coat protein, however internalisation is more selective. (Virus yoga: the role of flexibility in virus host cell recognition. Trends Microbiol. 2004 12:162-169).

PENETRATION involves phagocytosis into phagocytic vacuoles, after which the toxic activity of the pentons is responsible for rupture of the phagocytic membrane and release of the particle into the cytoplasm.

Uncoating follows an ordered sequence, first the pentons, releasing a spherical, partially uncoated particle into the cytoplasm. The core migrates to the nucleus where the DNA enters through nuclear pores, whereupon it is converted into a virus DNA-cell histone complex.

Gene Expression:

Before and independently of genome replication, immediate early and early mRNAs are transcribed from the input DNA. Transcription of the Adenovirus genome is regulated by virus-encoded trans-acting regulatory factors. Products of the immediate early genes regulate expression of the early genes. Early genes are encoded at various locations on both strands of the DNA (l = "leftward strand" and r = "rightward strand"). Multiple protein products are made from each gene by alternative splicing of mRNA transcripts - splicing was first discovered in Adenoviruses (Sharp, 1977).
Flint, J. and Shenk, T. (1997) Viral transactivating proteins. Ann. Rev. Genet. 31: 177-212.

The first mRNA/protein to be made (~1h after infection) is E1A. This protein is a trans-acting transcriptional regulatory factor whose precise mode of action is not known (not a DNA-binding "transcription factor") but is necessary for transcriptional activation of early genes. The protein is also capable of activating transcription from a variety of other viral and cellular promoters and shows no sequence-specificity, rather a modification of the cellular environment.
The second protein made is E1B. E1A + E1B together (and independently of other virus proteins) are capable of transforming primary cells in vitro (especially Ad5, Ad12).

Transformation is: "A CHANGE IN THE MORPHOLOGICAL, BIOCHEMICAL OR GROWTH PARAMETERS OF THE CELL", which may or may not result in cells which are able to produce tumours in experimental animals ( = NEOPLASTIC transformation).

The activities of the two proteins can be dissected out by molecular techniques:

E1A: Can immortalize primary cells in vitro.

E1B: Does not transform cells on its own, but "co-operates" with E1A to stably transform cells.

E1A + E1B: Necessary for full transformation and tumour formation in animals.

E1A binds to a cellular protein, p105-RB, the product of the retinoblastoma gene (retinoblastomas result when this gene is deleted or damaged, hence it is an "anti-oncogene" or "tumour suppressor"). The retinoblastoma susceptibility gene product, pRb, and related proteins (p107 and p130) form complexes with a transcription factor, E2F, that are dissociated by E1A to release transcriptionally active E2F. Expression of E2F can stimulate DNA synthesis in quiescent cells. It has also been established that other host cell cycle control proteins, e.g. cyclin A, bind to CR2. E1A proteins can bind p300 and the pRb related proteins simultaneously, thus potentially facilitating their interaction. Loss of Rb function also results in the induction of p53, by increasing the expression of the tumour suppressor protein p14ARF, which prevents MDM2 controlled degradation of p53. This is one of the mechanisms by which E1A causes apoptosis in primary cells.

• Why do some virus infections induce apoptosis?

E1B binds to p53, another tumour suppressor involved in the control of the cell cycle. N.B. Binding of DNA virus nuclear proteins to cellular tumour suppressors is a shared mechanism of cell transformation found in several virus families. The E1B region encodes two proteins of 19 kDa and 58 kDa that are unrelated in amino-acid sequence, but both have anti-apoptotic function. These two proteins act independently and by different mechanisms. The E1B 58 kDa protein directly abolishes the activity of the tumour suppressor, p53, the most commonly mutated protein in human cancer. The p53 protein is a transcription factor that negatively regulates cell proliferation. It is thought that p53 is not constitutively involved in the cell cycle, but is activated in response to DNA damage. When p53 is in a complex with E1B-58K protein, it is unable to transactivate transcription. Mutants of E1B-58K that are unable to form a complex with p53 are also unable to transform cells in culture in cooperation with E1A. The E1B 19-kDa protein inhibits apoptosis by a mechanism similar to that of human bcl-2 protein.

• Why should a virus want to inhibit apoptosis?

Adenoviruses also encode several other proteins that modulate immune-mediated apoptotic mechanisms. Most of these are derived from the E3 transcription unit. There are seven E3 proteins, none of which is required for replication in cultured cells, implying anti-immune functions. E3-gp19K (a 19 kDa glycoprotein coded by the E3 transcription unit) is the first line of defense against CTL, binds to all haplotypes of human class I antigens and is conserved in all respiratory adenoviruses. Adenoviruses which infect the gastrointestinal tract lack this protein, but they downregulate class I molecules by repressing at the transcriptional level, repression is meditated by the Ad-coded E1A proteins. Other E3-coded proteins named RID and E3-14.7K inhibit some of the killing pathways induced in infected cells by CTL, those that involve apoptosis rather than the perforin-granzyme pathway. E3 has therefore been called the "stealth" gene, allowing adenoviruses to evade the host immune response.

Together, these observations indicate that Adenoviruses, in the course of sequestering cellular machinery and altering the intracellular environment to favour viral replication, have profound effects on cellular functions. Viewed in this light, transformation is just an accidental (and rare) outcome of infection. The basis for oncogenesis (c.f. immortalization of cells in vitro - above) is not clear, but it is known that Ad12 E1A turns off class I MHC expression, possibly allowing tumours to escape destruction by CTLs.

DNA Replication:

Adenovirus DNA replication has been studied extensively both in vivo (t.s. mutants in infected cells) and in vitro (nuclear extracts). At least 3 virus-encoded proteins are known to be involved in DNA replication:

• TP (a.k.a. Ad DNA Pro) acts as a primer for initiation of synthesis.
• Ad DBP - a DNA-binding protein.
• Ad DNA Pol - 140kD DNA-dependent polymerase.

The steps involved in adenovirus DNA replication can be summarized as follows:

• First, the viral genome is coated with DBP.
• This protein reacts co-operatively with the cellular transcription factor, NFI which binds to a recognition site within the origin of replication, separated from the 1-18 bp core by a precisely defined spacer region.
• NFIII also binds at a specific recognition site between nucleotides 39 and 48.
• Protein-protein interactions, between NFI and pol, and pTP and NFIII help recruit the pTP-pol heterodimer into the preinitiation complex.
• Interaction between the heterodimer and specific base pairs 9 to 18 in the DNA sequence ensures correct positioning and the complex is further stabilized by interactions between the incoming pTP-pol and the genome-bound TP.
• DNA replication is then initiated by a protein priming mechanism in which a covalent linkage is formed between the alpha-phosphoryl group of the terminal residue, dCMP and the beta-hydroxyl group of a serine residue in pTP, a reaction catalysed by pol. This acts as a primer for synthesis of the nascent strand.
• Base pairing with the second GTA triplet of the template strand guides the synthesis of a pTP-trinucleotide, which then jumps back 3 bases, to base pair with the first triplet (also GTA) and synthesis then proceeds by displacing the non-template strand.
• NFI dissociates as the first nucleotide binds just prior to the initiation reaction. Dissociation of pTP from pol begins as the pTP-trinucleotide is formed and is almost complete by the time 7 nucleotides have been synthesized.
• NFIII dissociates as the replication binding fork passes through the NFIII binding site.

Late Transcription:

Pathogenesis:

Note that this list includes relatively common infections (at the top) and some rare infections (bottom). Most Adenovirus infections involve either the respiratory or gastrointestinal tracts or the eye.

Adenovirus infections are very common, most are asymptomatic. Most people have been infected with at least 1 type at age 15. Virus can be isolated from the majority of tonsils/adenoids surgically removed, indicating latent infections. It is not known how long the virus can persist in the body, or whether it is capable of reactivation after long periods, causing disease (it is hard to isolate this occult virus as it may be present in only a few cells). It is known that virus is reactivated during immunosuppression, e.g. in AIDS and transplant patients. Pre-existing adenovirus infection is a major problem in the rejection of transplanted hearts and lungs: Shirali GS, et al. N Engl J Med 2001;344:1498-1503,1545-1547.

A characteristic feature of adenovirus infection is their ability to subvert the host immune response by using the early E3 cassette of genes (Immune evasion by adenovirus E3 proteins: exploitation of intracellular trafficking pathways. 2004 Curr Top Microbiol Immunol 273, 29–85). The E3 membrane proteins are able to downregulate critical recognition structures for the host immune system from the cell surface. The molecular mechanism for removal depends on the E3 protein involved: E3/19K prevents expression of newly synthesized MHC molecules by inhibition of ER export, whereas E3/10.4-14.5K down-regulate apoptosis receptors by rerouting them into lysosomes.

Therapy:

None. Inactivated vaccines have been developed and are routinely used for military recruits in some countries (notably the USA). This is because adolescents and others in close daily contact are at risk for epidemic spread of respiratory infections - risk to general population is so low that vaccination is not a viable proposition.
Beginning in 1971, all US military recruits were vaccinated against adenovirus, but the sole manufacturer of the vaccine halted production in 1996. Since 1999, approximately 10% to 12% of all recruits have become ill with adenovirus infection in basic training and several death have been attributed to adenovirus infection. New vaccine lots are in preparation but will not be available for several years.

In recent years, there has been considerable interest in developing Adenoviruses as defective vectors to carry and express foreign genes for therapeutic purposes. One reason for this is that the Adenovirus genome is relatively easily manipulated in vitro (c.f. Retroviruses) and the genes coupled to the late promoter are efficiently expressed in large amounts.

MicrobiologyBytes: Adenovirus vectors - new genes, new vaccines

• The adenovirus genome is transcribed in both directions and alternative splicing allows multiple transcripts to be generated from the same sequence. Any manipulation of the virus genome therefore requires the coding capacity of both DNA strands to be considered.
• The adenovirus particle limits the amount of DNA that can be packaged to ~105% of the genome, only ~1.8kb more that the wild-type virus genome.
• The E3 gene region is dispensable for adenovirus replication in vitro and can be deleted from both replication-competent and replication-deficient vectors to permit larger inserts.

Vector systems have been developed in which most or all adenovirus protein coding sequences are removed. From the viewpoint of safety, size of transgene insertion and absence of vector gene expression, these so-called 'gutless' adenovirus vectors are attractive. They contain the inverted terminal repeats (ITRs) and adjacent sequences essential for replicating the adenovirus genome, including the cis-acting signal sequence (psi) which directs packaging of adenovirus DNA into virus particles. The gutless vectors require virtually all adenovirus gene functions to be provided for vector propagation and, since this cannot yet be achieved using a packaging cell line, they must be provided using a helper cell. Although their potential is enormous, current gutless vectors are complex and technically difficult to use. However, there are dangers associated with the use of adenovirus vectors, and a few deaths have occurred in clinical trials.

Adenoviruses: update on structure and function. J Gen Virol. 2009 90(1): 1-20

Update on adenovirus and its vectors. J. Gen. Virol. (2000), 81: 2573-2604.

Roulston, A. et al. (1999) Viruses and apoptosis. Ann. Rev. Microbiol. 53: 577-628

Yewdell, J.W. and Bennink, J.R. (1999) Mechanisms of Viral Interference With MHC Class I Antigen Processing and Presentation. Ann. Rev. Cell. Dev. Biol. 15: 579-606.

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