Genome structure of incomplete particles of adenovirus

Abstract

Incomplete particles arising during productive growth of adenovirus were separated from infectious particles by density gradient centrifugation. The DNA contained in particles of low density was characterized by restriction enzyme analysis and by electron microscopy and heteroduplexing techniques. The DNA is heterogeneous in length, ranging in size from 15% of the normal genome to full length. Many individual molecules contain long, inverted terminal repetitions, which consist of the sequences extending from the normal left-hand end of the viral genome inward; the normal right end sequences appear to be missing from these molecules. The region of the genome reiterated in these molecules is that which has been implicated in transformation of rat cells by adenovirus (Gallimore, Sharp, and Sambrook, 1974; Graham, van der Eb, and Heijneker, 1974). A model for adenovirus replication is presented that accounts for the aberrant structures observed.

Segregation of viral double‐stranded and single‐stranded DNA molecules in nucleo fo adenovirus infected cells as revealed by electron microscope in situ hybridization

“…Those of the viral nucleoids which were labeled when hybridization was performed on non-denatured sections previously digested by protease and RNase to reveal ssDNA, probably result from the beginning of insertion of replicating molecules. An eventual cessation of replication could lead to the formation of defective particles, the genome of which can be shorter than the entire genome [1,2,19]. This hypothesis, however, seems unlikely since defective particles are rare in human cells infected with Ad5 at a normal multiplicity of infection [3].…”

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

“…Tolun andPettersson, 1975;Ariga and Shimojo, 1977;Lechner and Kelly, 1977;Sussenbach and Kuijk, 1977). Copying of the other strand could occur by one of three processes: (i) a fully displaced strand can form a panhandle structure by hybridization of the bases within the ITRs and such a structure, being identical to the ends of duplex DNA, could initiate replication by the same mechanism (Daniell, 1976); (ii) initiation may take place off single-stranded templates; and (iii) a molecule which has undergone initiation at one end may initiate at the opposite end before the replication fork has progressed the full length of the genome (Lechner and Kelly, 1977). Currently there are no data which discriminate between these altematives and any or all of them may be operating during viral DNA replication.…”

Control of adenovirus packaging

“…1, Step 1). Ad DNA is packaged in a polar fashion starting at the left end of the genome [Daniell, 1976; Tibbetts, 1977; Hammarskjold and Winberg, 1980; Hearing et al, 1987]. Extensive analyses of the Ad5 genome have shown that there is a domain between left end nucleotides 200 and 400 that is absolutely required for encapsidation of viral DNA [Hearing et al, 1987; Grable and Hearing, 1990; Grable and Hearing, 1992; Schmid and Hearing, 1997, 1998, 1999; Sandig et al, 2000; Ostapchuk and Hearing, 2003b].…”