In this study, we purified the KSHV DNA binding protein ORF6 and examined its physical state using TEM and gel filtration. The protein was expressed and purified from insect cells. Size exclusion chromatography demonstrated that it exists as a heterogeneous mixture of small oligomers ranging in size from monomers to what are likely to be tetramers and then very large species beyond the Superdex 200 resolution limit. EM analysis revealed both species, with the smallest particles having a size expected for monomers. The very large molecular weight material was present in the form of long protein filaments, which form in the absence of DNA. Using a gentle EM preparative method involving rapid freezing and freeze-drying, images of the filaments were obtained that support a model in which the filament consists of two protein chains wrapped about each other.
Formation of the long ORF6 filaments required prolonged incubations, however less ordered filaments were seen after a few hours of incubation. While HSV-1 ICP8 and KSHV ORF6 both form these long protein filaments, ICP8 filamentation requires magnesium while ORF6 filamentation does not and while reducing agents were essential for consistent ORF6 filament assembly they are not needed for ICP8.
Whether these filaments are also engaged in an active dissociation into monomers and small oligomers was unclear, however incubation at 4 °C was seen to push the distribution toward smaller forms arguing that the filaments will dissociate. EM inspection did not reveal a significant number of short protein filaments in the mixtures, which are seen in preparations of RecA protein [26
]. With RecA, these short polymers were presumed to nucleate the formation of much longer filaments [26
]. If the ORF6 filaments dissociate or grow from the filament ends, then the circular species in which two ends have fused would be blocked for further elongation or dissociation and one would expect to see a build-up of circles over time. Future studies using other physical methods will be needed to probe the nature and kinetics of filament formation.
The filament formed by ICP8 in the absence of DNA measures 18 nm in diameter with a 25 nm helical repeat [22
]. It was also determined to be a two-start left handed filament formed by two protein chains wrapping about each other [22
]. We measured the ORF6 filament to be 14 nm in diameter with a longer, 43 nm helical repeat. We have not yet assigned handedness for the ORF6 filament. Since ORF6 has higher sequence similarity to BALF2 than ICP8, it will be interesting in the future to determine if full length Balf2 protein is able to form filaments in the absence of DNA. In a previous study [22
] we found that neither the C terminal truncated form of ICP8 nor the C terminal truncated Balf2 protein could form protein-only filaments, but both do bind single strand DNA.
ORF6 and ICP8 form long helical protein filaments in the absence of DNA, and for ICP8, the filaments are similar to those formed along single-stranded DNA. It has generally been assumed that in vivo, the DNA binding proteins polymerize from a pool of free monomers onto the DNA to generate the helical filaments that catalyze strand annealing and recombination. However in the case of KSHV, for example, the high local concentration of ORF6 in the nuclear replication bodies raises an alternative scenario. Here self-assembled ORF6 filaments are proposed to be present in the replication foci where they may provide a structural scaffold upon which other viral and host proteins involved in replication and recombination might bind. Rather than assembling filaments de novo along the viral DNA, DNA may meld into the pre-assembled filaments, an event which could greatly speed steps of strand annealing and recombination. This is a very different perspective on how these proteins would catalyze DNA transactions from the classic view in which the proteins assemble from a pool of free monomers. Further experiments will be required to probe this hypothesis.