Electron microscopy study of viral DNA packaging with lambda head mutants

Abstract

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA −-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In λNu1 −- infected cells, however, most petit λ was not bound to DNA. In Fec − cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In D − mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI −-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein p Nu1 is required for complex formation between head precursors and DNA concatemers, pA for the initiation of DNA packaging, pD and pF I for the promotion of DNA packaging, and pD for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.