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closeL2 disulfide with L2 or ?
Posted by Hood2031 on 21 Mar 2009 at 15:44 GMT
This work brings up very interesting questions about the structure of HPV capsids. For instance, I wonder if it is possible that the L2 homodimer observed under nondenaturing conditions may actually be a L2 heterodimer/trimer complex with another protein (i.e. L1?). The current cryo-EM structure of HPV16 PsV suggests that L2 N-termini stick out through the center of each L1 pentamer. Does this work suggest that in C22S and C28S mutant particles, the external loop collapses within the virion and forms dimers below L1 pentamers? This would be a gross structural rearrangement due to a very subtle amino acid substitution.
RE: L2 disulfide with L2 or ?
skcampos replied to Hood2031 on 23 Mar 2009 at 19:21 GMT
Hello, Thanks for your comments. Glad to discuss the paper with you.
We interpreted the ~150 kDa bands observed with the single cysteine mutants in non-reducing SDS-PAGE in Fig. 2C as disulfide-linked L2 homodimers for two main reasons. First, we excluded the possibility of an L2-L1 complex because the corresponding bands are not observed in the anti-[L1] blot, even after very long overexposure (not shown in paper). Second, Chris Buck's paper on the cryoEM localization of L2 (Buck et al. 2008, J Virol 82(11): 5190-5197) included some nice bimolecular fluorescence complementation data with split YFP fused to L2. The data suggested that the N-terminal ends of separate L2 molecules can be closely positioned (close enough to enable complementation of split YFP fragments) within the HPV virion. It is possible that these 150 kDa bands could be L2 linked to some other ~75 kDa protein but we feel they likely represent L2-L2 dimers.
Regarding the current model of L2 within virions, I believe its generally accepted that the central cavity of the L1 pentamers is too small for L2 to squeeze through and L2 likely reaches the surface of the virion by extruding between neighboring L1 pentamers (Sapp and Day, 2009, Virology 384(2): 400-409). While regions towards the N-terminus of L2 have been shown to be surface-exposed on virions, it's thought that the extreme N-terminal region, including the 9-12 furin cleavage site and the 17-36 RG-1 epitope are buried within the virion until conformational changes cause their exposure. The data presented in this paper suggest a buried intramolecular L2 disulfide and support these models of a buried N-terminus.