Adviser:Dr.Hosin Keyvani

Sherko Naseri

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Today we discuss about that how adenovirus, a DNA

virus, recruits cellular and viral factors and makes use of

its own cysteine protease to regulate capsid assembly…

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With the discovery of the genetic code it was recognised

that any given viral genome was too small to encode a

single polypeptide making up the entire capsid. It was

found instead, that a capsid was constructed of one or

several smaller polypeptides arranged as symmetric

building elements

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Capsid formation may be assisted by scaffolding

structures or cellular chaperones, both of which are not

parts of the final capsid structure

Packaging of the nucleic acid can occur by at least three

different mechanisms.

(I) A proteinaceous capsid is built around a condensed

nucleic acid,

(II) a capsid shell is built and the nucleic acid

packed and condensed within the shell

(III) the nucleic acid is condensed sequentially within the

growing capsid

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Directionality of assembly can also be conferred by

limited proteolysis of capsid proteins. These reactions

are catalysed by either host or virally encoded enzymes

and may stabilise or destabilise the capsid depending on

the virus. In the case of enveloped viruses, proteolytic

processing of surface spike proteins also confers

additional functions, such as the ability to fuse

membranes

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Protein IIIa links adjacent facets by spanning the capsid

Protein IX stabilises groups of nine trimeric hexons

protein VI anchors the ring of peripentonal hexons by

bridging to the DNA core inside the capsid

Other minor proteins, such as protein VIII, X, XI or XII

have not been localised unequivocally but are likely to

occur at the inside of the capsid

The DNA is condensed with proteins V, VII and the minor

component µ and covalently linked to two terminal

proteins sitting at each a 5' end.

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Also inside the adenovirus capsid are 10 to 50

copies of the cysteine protease p23.

the cysteine protease L3/p23, located in the

internal cavity at ~10 copies per virion

This protease, encoded in the late cassette L3

as a 23 kDa polypeptide, is essential during the

assembly of the virus in the nucleus of infected

cells (Weber, 1995).

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L3/p23 has eight cysteine residues, of which at least one is needed for proteolyticactivity (Weber, 1995). A thiol-disulfide exchange mechanism is thought to provide activation of the protease during virus assembly via a disulfide-linked peptide dimer corresponding to the C terminal peptide of the protein VI precursor (Mangelet al.,1993; Webster et al., 1993).

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The first step to assembly is the formation of hexon,penton base and fibre capsomers. While fibre and penton base oligomerisations occur independently of chaperones, hexon trimers are formed in the cytoplasm by a transient,directassociation of nascent polypeptide with a chaperone of Mr 100 000 in a 1:1 complex. This assisted folding pathway is thought to help to avoid assembly errors. A completely folded hexon trimeris then imported into the nucleus perhaps in association with the precursor of protein VI

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Empty capsids have a characteristic buoyant density of

1·31 g/mL and are made up of hexon, penton base, fibre,

and precursors of proteins IIIa, VI and VIII, but lack DNA.

Empty capsids also contain scaffolding proteins L1

52/55K and perhaps minor proteins of unknown function

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the late region 1 (L1), such as the scaffolding proteins of

Mr 52 000 and 55 000, respectively, and the precursor of

protein IIIa, are involved in DNA encapsidation

It is also clear that packaging somehow depends on a

cis-acting DNA sequence containing AT-rich repeats at

the left end and trans-acting in the right of the viral

genome.

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Possibly, specific trans-acting packaging factors

recognise this and adjacent DNA regions and thus

facilitate polar encapsidation of viral DNA into empty

capsids. Whether specific proteins are added to the

capsid once it is filled with DNA in order to lock-in the

DNA is unknown

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mutant viruses that lack the functional protease (tsl)

failed at releasing fibers and penetrating into the cytosol.

The mutation in ts1 responsible for the lack of processing

has been mapped to a C-T transition resulting in a

proline to leucine exchange at position 137 of the p23

proteinase

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They are most prominent in the ts1 mutant at the

restrictive temperature and arise due to the lack of

functional protease p23. ‘Young virions’ have the same

specific density as wild particles (1·34 g/mL), but lack

processing of precursor polypeptides pIIIa, pVI, pVII,

pVIII, pµ and preterminal protein (pTP) and are devoid of

polypeptides X, XI and XII and mr55000 scaffolding

protein

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The first cofactor identified has been the 11 amino acid

C-terminal peptide of the precursor to protein VI, pVI.

This activating peptide contains a critical cysteine residue

at the C-terminal position -2, which engages in a

disulphide exchange reaction with cysteine 104 of p23 as

indicated by biochemical studies and the crystal structure

of p23 bound to the activator peptide.

Possibly, a second activator of p23, polyanions, including

DNA, either stimulate p23 activity or stabilise the

enzyme.

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It is possible that p23 enters the capsid, perhaps in

association with the viral DNA as a complex with proteins V

and VII

This interaction would directly or indirectly lead to N- and C-

terminal cleavage of pVI and the production of the activating

peptide.

This could be part of a conformational change enabling the

fully processed pVI to stably lock-in with hexon

In agreement with such a scenario, blot overlay experiments

have detected an interaction between hexon and processed

pVI, but not with the precursor of VI.

How the scaffolding proteins exit the capsid is unknown. It is

possible that they leave through openings in an incomplete

capsid, or via transient holes created by conformational

changes in the shell, analogous to bacteriophage maturation

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Lytic release of adenovirus is facilitated by a virally

encoded Mr 11 600 polypeptide also called ADP

(adenovirus death protein) of the delayed early

transcription unit E3.

Whether wild type p23 protease, which cleaves parts of

the cytoplasmic intermediate filament network, directly or

indirectly facilitates virus exit rather than cell lysis is not

known

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The role of the adenovirus protease in virus entry into cells Urs F.Greberl'2, Paul Webster, Joseph Weber3 and Ari Helenius

Virus Assembly and Disassembly: the Adenovirus Cysteine Protease as a Trigger FactorUrs F. Greber

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