STRUCTURAL BIOLOGY OF VIRUS ASSEMBLY

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This project aims to elucidate the molecular mechanisms that control the assembly of viral capsids with the twin goals of defining prospective targets for antiviral compounds and gaining insights into regulation of the assembly of macromolecular complexes in general. Our major results for the past year are: (i) Evidence is accruing that some capsids serve as functional compartments in which specific genome-related processes take place, i.e. they are not simply closed containers to which viral genomes are confined. For hepatitis B virus (HBV), the RNA pregenome is retrotranscribed inside the capsid, which accordingly, is fenetrated with about 20 A holes that allow the entry of nucleotides and egress of digested RNA. The L-A virus capsid serves also operates as a biosynthetic chamber: in this case, transcripts are fed out into the cytoplasm through holes large enough to admit ssRNA but small enough to prevent egress of the dsRNA template. (ii) We completed and published the structure of the HBV capsid at the unprecedentedly high resolution (for electron microscopy) of 9 A, developing computational methods that should aid work on other viruses and macromolecular complexes. A comparably detailed analysis was also accomplished for bovine papillovavirus, visualizing its helical inter-capsomer linkers. For HBV, the resolution achieved was sufficient to visualize the a-helices that make up about half of the 150-residue assembly domain, but not high enough to allow tracing of the chain. Progress towards the latter goal was made by precisely localizing its C-terminus by appending a cysteine at position 150, binding an 11-atom gold cluster to it, and visualizing the cluster by cryo-EM. The C-termini congregate together under all 5-fold and 6-fold axes. This observation provides a "molecular crowding" explanation for how this peptide operates as the morphogenetic switch that we previously found to specify whether more large (T=4) or small (T=3) capsids are assembled. The success of this experiment suggests that we may be able to map other residues in the same way, ultimately defining the protein fold. (iii) Encapsidated bacteriophage T7 dsDNA was shown to be packed as a tightly wound coaxial spool by a combination of cryo-EM with computational modeling, taking advantage of a mutant with the serendipitous property of providing two well defined, mutually perpendicular, views when observed in thin ice films. This result represents the first conclusive determination of the three-dimensional structure of a condensed (quiescent) chromosome of any kind. T7 DNA is packed remarkably tightly, with a spacing between neighboring B-form duplexes of only 25 A, center-to-center, matching that in a hexagonal phase crystal. In contrast, the dsRNA of L-A is most likely A-form, to judge by our measurements of mass-per-unit-length by STEM microscopy and it is packed much more loosely (about 40 A, center-to-center). This loose packing allows for the churning motion that the genome must undergo within the L-A capsid as it is propelled past the immobilized polymerase during replication and transcription.