Bipartite structure and functional independence of adenovirus type 5 packaging elements

Abstract

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.

Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains

“…The same viruses package with equal efficiency in individual, single-virus infections. Similar results are observed in coinfection experiments using wild-type Ad5 and packaging domain mutants: a greater reduction in packaging efficiency of mutant viruses is found in coinfections than in single infections with the same viruses (12,13,26). These observations are consistent with the model that competition for a limiting trans-acting component occurs within an infected cell.…”

“…Determination of virus yield and packaging efficiency. Both assays were performed as described previously (26). For the determination of virus yield in a single infection, infected cell lysates were prepared 48 h postinfection and the amount of infectious virus was determined by plaque assays on 293 cells.…”

“…The Ad5 packaging domain consists of at least seven redundant, albeit not functionally equivalent, elements termed A repeats I through VII (AI through AVII) (12,13). We have previously shown that the four most dominant A repeats, I, II, V, and VI, exhibit a bipartite consensus motif 5Ј-TTTGN 8 CG-3Ј, which is conserved across a number of Ad serotypes (26). These four repeats are aligned relative to each other in Fig.…”

“…This decrease in virus growth is thought to reflect the competition of limiting, trans-acting packaging components from viral ge-nomes by the unlinked packaging domain fragments, resulting in their inability to be encapsidated. Additionally, in coinfection experiments, viruses carrying more A repeats package with greater efficiency than viruses with fewer A repeats even though comparable levels of nuclear viral DNA are available for encapsidation with both viral genomes (26). The same viruses package with equal efficiency in individual, single-virus infections.…”

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].…”