Structural Biology Of Virus Assembly

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

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Abstract

Summary: Our interest in virus assembly is twofold. We seek to identify features with potential for antiviral drug and vaccine interventions, and to elucidate mechanisms at play in the assembly, maturation, and activation of large macromolecular complexes in general. In this context, we work on several viral systems with particular focus on viruses with genomes of double-stranded DNA. These viruses have the largest viral genomes and correspondingly elaborate virion structures. We also have a major interest in hepatitis B virus, a major human pathogen, and in retroviruses, primarily HIV-1. The latter (HIV-related studies) is the subject of a separate report (AR041166-11). During FY18, we made progress on several subprojects: 1) Herpesvirus assembly. Over the past 25 years, we have studied many aspects of herpesvirus assembly, mostly on herpes simplex virus type 1(HSV-1). In recent years, our focus shifted to nuclear egress. The HSV-1 capsid assembles in the nucleus, then migrates into the cytoplasm for subsequent steps on the pathway. The Primary Enveloped Virion (PEV) is a transient particle formed in the perinuclear space as the DNA-filled capsid traverses the nuclear envelope. As the capsid buds through the inner nuclear membrane, it becomes coated with nuclear egress complex (NEC) protein. This yields a PEV whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. To obtain enough PEVs for structural analysis, we isolated them from US3-null-infected cells (pUS3 is a virally encoded kinase). We found that PEVs differ from mature extracellular virions (MEVs) in several respects. PEVs have very few glycoprotein spikes whereas MEVs are densely coated with them. PEVs are 20% smaller than MEVs and there is little space between the capsid and the NEC layer, whereas in an MEV, this space is more capacious and is occupied by the tegument. In FY18, we sought to further characterize the portal vertex. To do so, we worked with A-capsids (empty capsids in a mature conformational state), using a UL25-null strain which yields a high percentage of A-capsids. Tomographic data were recorded and, using a processing algorithm developed in the LSBR, it was possible to identify the portal vertex on these capsids. The portal protein pUL6 is seen to be embedded in the capsid floor of the capsid. In these reconstructions, we also detected a sizeable density overlying the portal vertex that may represent the viral terminase complex. 2) Packing of DNA and internal proteins in bacteriophage T4. In earlier work, our cryo-EM studies of bacteriophage T7 provided strong evidence for the concentric spool model whereby the DNA is coiled around the portal axis in nested shells (Cerritelli et al., Cell 91, 271-290 1997). We have now investigated whether a similar arrangement pertains in T4, which has a 4-fold larger genome, a prolate capsid, and lacks the cylindrical protein core of T7. Cryo-EM yielded side-views and axial views (defined as such relative to the axis running through the portal vertex) of prolate (wild-type) heads and of isometric (mutant) heads. Giant heads were imaged in sideview. We conclude that in isometric heads, the DNA is spooled around the portal axis, essentially as in T7; in giant heads, the spool is rotated by 90o. The innermost regions of DNA-filled heads are less ordered. We infer that the conformations of encapsidated genomes represent an energy-minimized compromise between electrostatic repulsion effects involving DNA duplexes and the strain associated with bending the DNA. Although T4 does not emulate T7 in having a highly ordered core of internal proteins, it does have substantial amounts of internal proteins. In both systems, these proteins are destined for delivery into a host cell whose locations in the head have been unclear. To investigate the internal proteins ofT4, of which there are two, gpAlt and gpIPIII, we have used bubblegram imaging, a technique invented in the LSBR (Wu et al., Science 335, 182 2012). In this way we found that both gpAlt and gpIPII are excluded from a highly ordered peripheral zone but otherwise are distributed seemingly at random throughout the capsid interior. The peripheral zone coincides with that occupied by shells of coiled DNA. 3) DNA packaging into supersized T=7 capsids of a thermophilic virus. This project has been a joint undertaking with F. Antson (University of York) as part of a Wellcome Trust/NIH collaborative program. Double-stranded DNA viruses including bacteriophages and herpesviruses package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded capsid can package DNA in the presence of large terminase and ATP. Cryo- EM reconstructions were determined to 0.4 nm resolution for both the expanded capsid and the procapsid. Unexpectedly, the capsid was found to observe T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of T=7 and other phages (e.g. HK97, P22) with the same T-number (7) in which DNA packing density is thought to approach the maximum physically possible. Our reconstructions explain this anomaly, whereby modifications to the canonical HK97 fold permit a capsid volume twice as large. We also obtained a 1.95 crystal structure for the portal protein and performed symmetry-free reconstructions for the procapsid and expanded capsid to define its setting in the portal vertex. We found that capsid expansion elicits a substantial change in the conformation of the portal protein, while still allowing DNA to be packaged. 4) Encapsulins are protein shells that resemble viral capsids in many respects, including the fold of their constituent subunits and the icosahedral symmetry of their molecular architecture. This fold was first observed in capsids of bacteriophage HK97. However, instead of housing genomic nucleic acid as in viral capsids, encapsulins accommodate other kinds of cargo. In a paper published in FY15, we described the structure and essential functional properties of an encapsulin of the Gram-negative bacterium Myxococcus xanthus. This particle has an icosahedral protein shell 32 nm in diameter and an icosahedral triangulation number of 3. Thus EncA is assembled from 180 copies of EncA protein; it also has smaller amounts of three internal cargo proteins (EncB; EncC; EncD). Native nanocompartments isolated from M. xanthus have dense iron-rich cores. Functionally, they resemble ferritins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). Their role appears to be to scavenge excess cytoplasmic iron in order to protect the bacterium from reactive oxygen species. In continuing studies on this system, our main thrust has been to seek high resolution cryo-EM data on the purified cargo protein ClpB. ClpB has a disordered C-terminal region but its N-terminal regions is folded and forms a decamer with D5 symmetry. In our current model for the complex, these decamers are envisaged to bind to specific sites on the inner surface of EncA shells whereby aligned channels through the respective walls of the EncA and the EncB shells allow iron and lesser amounts of phosphorus to enter the EncB shell where they are deposited. EncC has now also been purified and found also to be a decamer. This analysis is ongoing.