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1.6.1 The E4 region encodes 7 different proteins

Transcripts from the E4 region are subject to alternate splicing events, leading to the production of approximately 18 distinct mRNAs, which are predicted to encode 7 different proteins named Open Reading Frame 1 (Orf1), Orf2, Orf3, Orf4, Orf3/4, Orf6, Orf6/7 (Virtanen et al., 1984). All, except Orf3/4 have been demonstrated to exist in infected cells. To date, all of the adenoviruses sequenced appear to have an E4 region, with the exception of chicken embryo lethal orphan virus (CELO), which is an avian adenovirus (Chiocca et al., 1996).

1.6.2 The E4 region is required for efficient DNA replication, late gene expression and shutoff of synthesis of host cell proteins

Halbert et al confirmed the requirement of early region 4 for efficient DNA replication, late gene expression and shutoff of synthesis of host cell proteins by generating mutant Ad5 viruses carrying defined lesions in the E4 region. Mutant virusdl366, which lacked the majority of the E4 region was severely defective,

and could only be propagated on stable Vero (monkey kidney) cells expressing the E4 region. Virusdl355, which lacked 14bp within the segment encoding the E4 Orf6 protein, showed a delayed onset of viral DNA synthesis. Expression of late viral proteins was reduced in bothdl355 anddl366, although more

severely indl366. Shutoff of host protein synthesis was also less efficient with the mutant viruses, more so with virusdl366 (Halbert et al., 1985).

Attempts to identify the specific role of each of the E4 proteins, by mutational analysis of each of the E4 Orfs, have been unsuccessful. This was due to the fact that only a mutation in the E4 Orf6 region demonstrated any phenotypic effect on virus growth, and this effect was minimal compared to deletion of the entire E4 region.

1.6.3 E4 Orf1

The E4 Orf1 protein, in a variety of different adenovirus serotypes, is thought to be involved in transformation, but only when expressed at sufficient levels (Javier, 1994; Ohman, 1995). Orf1 sequences appear to be related to dUTPase enzymes, although they lack an essential conserved dUTPase motif and are inactive in dUTPase assay (Weiss et al., 1997). Although it has been shown that the avian adenovirus CELO has a putative dUTPase gene in a location analogous to E4 Orf1 (Chiocca et al., 1996). The CELO E4 Orf1 gene contains the essential conserved dUTPase motif and is active in a dUTPase assay (Weiss et al., 1997). The role of Orf1 in lytic infection has yet to be determined. E4 Orf1 deficient Ad5 shows no growth defect in HeLa cells (Leppard, 1997).

1.6.4 E4 Orf2

There is at present no functional information regarding the E4 Orf2 protein, although it has been shown to be localised to the cytoplasm in infected HeLas, is produced at early times during adenovirus infection, and is not detected in a complex with other proteins (Dix and Leppard, 1995).

1.6.5 The E4 Orf3 and E4 Orf6 proteins can compensate for each other

The role of E4 Orf3 proteins and E4 Orf6 proteins has been discussed earlier in relation to E1B-55kDa and p53 proteins.

Bridge and Ketner demonstrated that the E4 products Orf3 and Orf6 could

compensate for each other. By generating a series of Ad5 mutants with deletions in the E4 region it was demonstrated viral late protein synthesis was essentially normal in viruses that were unable to express either Orf3 or Orf6. In mutants that could not express both Orf3 and Orf6, late protein synthesis was dramatically reduced and plaques formed with an efficiency less than 10-6that of wild type virus. Mutants that lacked Orf3 or Orf6 formed plaques with only slightly less efficiency than wild type virus (Bridge and Ketner, 1989).

Huang and Hearing also generated mutant Ad5 viruses in order to assign specific function to each of the E4 gene products. Mutant viruses which expressed Orf6 but lacked Orf3, behaved like wild type virus. Mutant viruses which expressed Orf3 but lacked Orf6 displayed a delay in the onset of viral replication, reduced levels of viral late protein synthesis and inefficient shutoff of host cell protein synthesis, and a 10 to 20 fold reduction in virus yield. Mutant viruses lacking both Orf3 and Orf6 displayed a significant lag in the onset of viral replication, a dramatically

reduced level of viral late protein synthesis, no obvious shutoff of host cell protein synthesis and up to a 105reduction in virus yield (Huang and Hearing, 1989).

Viruses that lack the E4 region show a number of severe phenotypes, including defects in viral mRNA accumulation, transcription, splicing, late protein synthesis, host cell shutoff and viral DNA replication. This defect is due in part to the

production of genome concatemers (Weiden and Ginsberg, 1994), caused by the covalent joining of viral genomes, and resulting in molecules that exceed the packaging capacity of the capsid. Concatemerisation is mediated by the host cell, through a Mre11/Rad50/Nbs1 complex (Boyer et al., 1999; Stracker et al., 2002). In Ad5 expression of E4 Orf6 and E1B-55kDa proteins results in the proteasome- mediated degradation of the Mre11 complex members (Stracker et al., 2002). The E4 Orf3 protein is able to exclude Mre11, Rad50 and Nbs1 from the viral

replication centres.

Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the

E1b55K/E4orf6 complex. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members (Stracker et al., 2005).

1.6.6 E4 Orf4

E4 Orf4 also regulates protein phosphorylation in the infected cell by binding to protein phosphatase 2A (PP2A) (Muller, 1992), resulting in the selective

hypophosphorylation of some proteins, including E1A. The hypophosphorylated E1A residues have been mapped, and at least one of these residues is a known target for mitogen-activated protein kinase (Whalen et al., 1997). Bondesson et al demonstrated that E4 Orf4 negatively regulates both E4 and E1A transcription, dependent on PP2A activity, and represses E1A-mediated activation of the E4 promoter (Bondesson et al., 1996). These data suggest the presence of a regulatory loop in which E1A activates expression of the E4 region. Expression of the E4 Orf4 protein results in the negative regulation of both E1A and E4 regions (Leppard, 1997).

Mannervik et al demonstrated that adenovirus E1A activation of the viral E2 promoter was abrogated by coexpression of the E4 Orf4 protein. The abrogation of E1A activation of the E2 promoter was deemed to occur through the E2F DNA binding sites present on the E2 promoter, demonstrated by the fact that E4 Orf4 inhibited E2F-1/DP-1 mediated transactivation, as well as E2 mRNA expression during virus growth (Mannervik et al., 1999).

1.6.7 E4 Orf3/4

This protein is predicted to exist, based on analysis of Ad2 mRNA structure in HeLa cells (Virtanen et al., 1984), although the protein has not yet been detected in infected cells.

1.6.8 E4 Orf6/7

E4 Orf6/7 dimerises to link two E2F molecules, thus facilitating binding at two E2F sites present in the Ad2 promoter, and activating transcription (Cress and Nevins, 1994; Huang and Hearing, 1989; Obert et al., 1994).

O’Connor and Hearing showed that the E4 Orf6/7 gene product induces binding of the cellular transcription factor E2F to the viral E2A promoter region, which is directly correlated with the transcriptional activation of the E2A promoterin vivo. The E4 Orf6/7 protein acts by functionally compensating for the E1A proteins. In the absence of E1A, expression of E4 Orf6/7 is sufficient to displace the

retinoblastoma protein family members from E2Fs, leaving E2Fs free to activate transcription of the E2A promoter (O'Connor and Hearing, 2000).