Capsid expansion follows the initiation of DNA packaging in bacteriophage T4

Most bacteriophages undergo a dramatic expansion of their capsids during morphogenesis. In phages lambda, T3, T7 and P22, it has been shown that expansion occurs during the packaging of DNA into the capsid. The terminase-DNA complex docks with the portal vertex of an unexpanded prohead and begins packaging. After some of the DNA has entered, the major head protein undergoes a conformational change that increases both the volume and stability of the capsid. In phage T4, the link between packaging and expansion has not been established. We explored the possibility of such a connection using a pulse-chase protocol and high resolution sucrose gradient analysis of capsid intermediates isolated from wild-type T4-infected cells. We show that the first particle appearing after the pulse is an unexpanded prohead, which can be isolated in vitro as the ESP (empty small particle). The next intermediate to appear is also unexpanded, but contains DNA. This new intermediate, the ISP (initiated small particle), can also be isolated on agarose gels, permitting confirmation of both its expansion state and DNA content (~ 10 kbp). It appears, therefore, that ≥ 8% of the T4 genome enters the head shell prior to expansion. Following packaging of an undetermined amount of DNA, the capsid expands, producing the ILP (initiated large particle), which is finally converted to a full head upon the completion of packaging. An expanded, empty prohead, the ELF (empty large particle), was also observed during 37°C infections, but failed to mature to phage during the chase. Thus the ELF is unlikely to be an intermediate in normal head assembly. We conclude by suggesting that studies on assembly benefit from an emphasis on the processes involved, rather than on the structural intermediates which accumulate if these processes are interrupted.