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

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

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

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 and covalently linked to two terminal proteins sitting at each a 5' end.

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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).

 L3/p23

has eight cysteine residues, of which at least one is needed for proteolytic activity (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 (Mangel et 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,direct association 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 trimer is 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 131 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 (134 g/mL), but lack processing of precursor polypeptides pIIIa, pVI, pVII, pVIII, p 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 Cterminal 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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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 Factor

Urs F. Greber

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