Deletion of the E4 region of the genome produces adenovirus DNA concatemers.

Abstract

Two mutants containing large deletions in the E4 region of the adenovirus genome H5dl366 (91.9-98.3 by replication-induced recombination (11). The last 45 bp of the genome contain the necessary sequence information for replication, and any deletion of the terminal cytosine abolishes DNA synthesis (12, 13).Other viral gene products may be involved in viral DNA synthesis, and other mechanisms not yet elucidated may be required for completion of viral DNA replication. Some phenomena not accounted for in the current model include the following: circular, supercoiled, and multimeric viral DNA molecules, which were identified in electron microscopic studies (14); the pL 5'-to-3' exonuclease required in the in vitro model (9) produces defective strands that need additional processing for maturation; a large proportion of viral particles produced during infection carry partially replicated single-stranded DNA (15); and concatemers of input DNA are formed in the in vivo plasmid model of viral DNA synthesis (10)(11)(12)(13). Topoisomerase, which is required in the in vitro model, should not be needed to relieve tortional stress in the linear molecules predicted by the current model. In addition, more than the three viral genes defined by the current model are needed for viral DNA replication, since mutations in the E4 region have a significant effect on viral DNA synthesis (16)(17)(18).To gain a better understanding of the functions of the E4 genes in adenovirus DNA synthesis, the DNA replication phenotypes of E4 mutants were investigated. These experiments revealed the formation of viral DNA concatemers, which suggests that multimeric genomes are a consequence of disruption of a normal viral gene function and that the mechanism of adenovirus DNA synthesis may differ from the presently accepted model. MATERIALS AND METHODS

Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

“…The membrane was washed for 1 hr at room temperature in 2x SSC/0.1% SDS and for 1 hr at 65°C in 0.2x SSC/0.1% SDS. The size of the E1A terminal HindIII fragment from a monomer genome is 2.8-kD in Ad5 and d1366" + ORF3, as shown in the Results (Weiden and Ginsberg 1994). The corresponding radioactive signal to E1A probe that hybridized to the filter was counted (PhosphorImager), and the values obtained are presented in the results as arbitrary units.…”

“…Thus, deletion of the E4 domain or selective deletion of these ORF3/ORF6 leads to a severe delay in the onset of adenovirus 5 (Ad5) replication as well as defects in the shut-off of the accumulation of host protein mRNA (Huang and Hearing 1989). In addition, ORF3/ORF6 mutants produce viral DNA concatemeres that lead to a "dead-end pathway" of viral replication (Weiden and Ginsberg 1994).…”

Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies

“…The DDR severely inhibits Ad DNA replication if unabated (13) because the ends of the viral genome are ligated to one another via the non-homologous end-joining pathway. This results in the loss of DNA sequences at the multimeric junctions (14,15), which contain the Ad origins of DNA replication. In addition, cytoplasmic sensors recognize dsDNA in the cytoplasm of infected cells early after virus infection and activate an IFN response (16).…”

E4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections.

“…These viruses produce concatemeric Ad DNA which includes a variety of DNA junctions between the two genome ends ; this is not seen in infections by wild-type or other types of E4-mutant virus (Weiden & Ginsberg, 1994). Such concatemers are not thought to be an essential feature of the Ad DNA replication mechanism.…”