Growing Pains of Bacteriophage Lambda: Examination of the Maturation of Procapsids into Capsids

The assembly of many complex double-stranded DNA viruses involves the insertion of viral DNA into a pre-formed procapsid structure. The procapsid assembly pathways are similar between the bacteriophage and eukaryotic viruses; in the case of bacteriophage lambda (lambda), the immature procapsid is assembled from 12 copies of the portal protein gpB, 10--12 copies of the gpC protein, the gpNu3 scaffold protein and the major capsid protein gpE. Current models suggest that procapsid maturation includes (i) a covalent fusion between the gpC and an equivalent number of gpE proteins, (ii) proteolysis of the newly fused protein to yield the minor capsid proteins pX1 and pX2, (iii) proteolysis of gpB to yield pB* and (iv) proteolysis and exit of gpNu3 from the procapsid shell.;The role that these maturation steps play in procapsid structure and/or function is unclear. Moreover, the protease responsible for procapsid processing is uncertain. In this work mutagenesis studies are presented that demonstrate gpC is the viral protease responsible for procapsid maturation. Biochemical studies show that proteolysis is not essential for the assembly or biological activity of the procapsids in vitro but is essential for late steps in virus assembly or infection. Unexpectedly, none of the putative gpC-gpE fusion product are detected in either wild-type or mutant procapsids. My studies demonstrate that assembly and maturation of the lambda procapsid is strongly conserved with the eukaryotic herpesviruses.;Packaging of the lambda genome into the procapsid triggers an expansion step yielding a more angular structure called the capsid, which is stabilized by the addition of the viral gpD protein. Also presented here are my in vitro studies of urea-triggered procapsid expansion. My results demonstrate that expansion is a cooperative two-state transition and is reversible when expanded capsids are transferred to a buffer containing greater than 1 mM Mg2+. The expanded state is stabilized by the addition of gpD even in the presence of Mg2+. Finally, my results demonstrate that the addition of gpD to capsids is not cooperative despite previous studies suggesting otherwise. Studies presented here could lead to the use of lambda procapsids for drug delivery.