Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of UL28 in this proc ess, purified U(L)28 protein was assayed for the ability to recognize conse rved herpesvirus DNA packaging sequences. We report that DNA fragments cont aining the pac1 DNA packaging motif can be induced by heat treatment to ado pt novel DNA conformations that migrate faster than the corresponding duple x in nondenaturing gels, Surprisingly, these novel DNA structures are high- affinity substrates for UL28 protein binding, whereas double-stranded DNA o f identical sequence composition is not recognized by U(L)28 protein. We de monstrate that only one strand of the pad motif is responsible for the form ation of novel DNA structures that are bound tightly and specifically by UL 28 protein, To determine the relevance of the observed U(L)28 protein-pad i nteraction to the cleavage and packaging process, we have analyzed the bind ing affinity of U(L)28 protein for pad mutants previously shown to be defic ient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with t he reported reduction in cleavage and packaging efficiency, thereby support ing a role for the U(L)28 protein-pad interaction in vivo. These data there fore suggest that the formation of novel DNA structures by the pad motif co nfers added specificity on recognition of DNA packaging sequences by the U( L)28-encoded component of the herpesvirus cleavage and packaging machinery.