BIPARTITE STRUCTURE AND FUNCTIONAL INDEPENDENCE OF ADENOVIRUS TYPE-5 PACKAGING ELEMENTS

Selectivity and polarity of adenovirus type 5 DNA packaging are believ ed 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 packagin g domain was mutationally dissected into at least seven functional ele ments called A repeats, These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with el ements I, II, V, and VI as the most critical repeats, Viral packaging was shown to be sensitive to spatial changes between individual A repe ats, To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separ ating A repeats I and II, as well as A repeats V and VI. The results o f our mutational analysis reveal previously unrecognized sequences tha t are critical for DNA encapsidation in vivo. On the basis of these re sults, we present a more complex consensus motif for the adenovirus pa ckaging 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 packa ging-deficient adenovirus: multimerization of packaging repeats is suf ficient for the evolution of packaging-competent viruses, Finally, we identify minimally sized segments of the adenovirus packaging domain t hat can confer viability and packaging activity to viruses carrying gr oss truncations within their left-end sequences, Coinfection experimen ts 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.