Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains

Abstract

Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of acis-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AI, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respectivecis-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting,trans-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.

Control of adenovirus packaging

“…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]. This domain can function at the right end of the genome as well, and recombinant genomes carrying packaging domains at both ends of the genome are viable [Hearing and Shenk, 1983; Parks and Graham, 1997].…”

“…Synthetic packaging A repeats were inserted into the Ad5 genome in place of the authentic packaging domain [Schmid and Hearing, 1998]. Viruses with WT or very close to wild type levels of packaging were recovered that contained: (1) a dimerized pair of A repeats 1 and 2; (2) dimeric copies of A repeats 5, 6, and 7; or (3) six tandem copies of A repeat 1.…”

“…These proteins may or may not have a dual role, one for transcription and another for packaging. Utilization of synthetic packaging elements as probes with in vitro DNA–protein binding assays narrowed the focus for relevant binding activities [Schmid and Hearing, 1998]. The cellular proteins OCT‐1 (octamer‐1) binding protein [Erturk et al, 2003], COUP‐TF (chicken ovalbumin upstream promoter transcription factor) [Cooney et al, 1992], and CDP (CAAT displacement protein) [Erturk et al, 2003], were found to bind the packaging elements along with the viral protein, IVa2 [Perez‐Romero et al, 2005], and an additional viral‐specific protein of unknown origin [Ostapchuk et al, 2005].…”

Xenogenic Adenoviral Vectors

“…For the xenogenic mastadenoviruses there are three ORFs at the left end that show homology with HAdVs [77][78][79][80]. The genome packaging signal is also present within the first ^500 nucleotides of the HAdV5 genome [81], but until recently this had not been defined for any xenogenic virus. For CAdV2, however, it was shown that the packaging region consists of a ^200bp region that contains redundant, but not functionally equivalent sequences.…”

“…For CAdV2, however, it was shown that the packaging region consists of a ^200bp region that contains redundant, but not functionally equivalent sequences. The consensus sequence for HAdVs [81] is present only once and is of minor importance [82].…”