Viral Assembly through Host Defense

1. Viral Assembly through Host Defense Kristin Shingler July 28, 2011

2. Modulation of T-Number P4 P2 P4 ProCapsid P4 gpSid

3. T7 vs T4 Conformation • P4 is a parasite of P2; produces NO structural proteins of its own • gpN is P2 major capsid protein; changes in flexibility of hexamers determine T number • gpSid (size-determining protein) binds to gpN; acts with gpO (scaffolding protein) altering hexamer angle, producing only T=4 conformation • Sir mutation prevents gpSid binding, producing only T=7 conformation; Sid is a brace crossing between 5-fold positions

4. HepB 7.4- and 9-A Reconstructions

5. Hepatitis B Virus Size Dimorphism • Full Length Capsid Protein produces T=3 & T=4 cores (13:1 Large:Small) • Truncating C-terminal tail of capsid protein results in loss of packaging and increase in number of T=3 cores • Cores aa 1-149 5% T=3, Cores aa 1-140 85% T=3, Cores aa 1-138 or less no assembly • Bulk of Assembly domain of HepBc protein is 4-Helix Bundle

6. Assembly of Big Viruses • Quasi-Equivalence is plausible for small T numbers, but less so as the number increases • Example: Algal Virus Paramecium bursariaChlorella Virus Type 1 T=169 1900-A diameter • Large complex viruses utilize scaffold and accessory proteins to aid in assembly

7. Adenoviruses

8. Difference Imaging • Identified non-hexon components and demonstrated their role in virion stabilization • Polypeptide IX  Trimers on top of hexons; form center of facet • Polypeptide IIIa  links facets across virion edge • Polypeptide VI  Links peripentonal hexons of adjacent facets inside the virion • Hexons are not distinct conformationally, rather their association with accessory proteins defines their role in viral assembly/structure

9. Herpes Simplex Virus-1 T=16

10. Procapsid Components • VP5  Major Capsid Protein • VP19c & VP23  Triplex Proteins • VP22a and/or pre-VP21  Scaffolding Protein • Triplexes exist at all 3-folds • Scaffolding protein may act as loose micelle so that VP5 can move on the surface and interact with triplexes. This defines interactions between capsomers, which survive to the mature virion.

11. Viral Genome Organization • Variety of genomes  ss, ds, RNA, DNA, (+), (-), linear, circular, host histones • 1D nature of genetic code renders all nucleocapsids asymmetric • Sections of genome adopt higher ordered structure • Icosahedral averaging renders asymmetric genome a featureless ball of mass in nucleocapsid

12. Cowpea Chlorotic Mottle Virus

13. CCMV Native vs. Swollen • Cryodensity map of swollen particles was used to fit native A,B,&C sub-units into and understand the structural changes necessary to convert between the two conformations. • Swollen CCMV RNA clusters at quasi 3-folds replacing native protein-protein interactions • This places RNA in a place to exit the capsid easily, and the RNA-protein interactions stabilize the expanded capsid

14. Flock House Virus

15. FHV • X-ray structure revealed regions of highly ordered duplex DNA (~20% Genome) in contact with inner capsid wall at 2-folds • Results duplicated in 22-A cryo reconstruction • GammaB & GammaC helicies contact bulk RNA close to cleavage site • C-terminus of GammaA helix contacts RNA with cleavage site 35-A away • Agrees with kinetics studies indicating 120 subunits cleaved faster than last 60 subunits

16. Release of Progeny Virus • Viruses that assemble in cytoplasm are released by cellular lysis. • Viruses that assembly on membranes are released by “budding”. • 2 Main Problems of Animal Cell Budding: •  Bud must form on CORRECT membrane •  Must incorporate viral proteins, while excluding host proteins

17. Alphavirus Budding

18. Alphaviruses • E1/E2 heterodimer formed in ER • p62 cleavage forms spike trimers • Spikes interact laterally via “skirt” domains • Spike transmembrane domain facilitates interaction with nucleocapsid • Binding is cooperative • Envelope proteins pack on membrane and form hexagonal arrays. The resulting lateral interactions exclude host membrane proteins, and produces a flat region which is stabilized by binding of the complementary capsid.

19. Virus Transmission • Little is known from cryoreconstructions • Small amounts of data suggest the virion structure adapts according to transmission requirements • Ex.  Alphavirus virion structure changes to replicate in 2 distinct hosts (arthropods and mammals) • Arthropod budding occurs in early secretory pathway versus mammalian budding through plasma membrane • Changes in timing of spike cleavage allow for this adaptation

20. Antibody Structure

21. Host Defense • Vertebrate Immune System is Complex • Innate and Adaptive Immunity • Viral neutralization by antibodies is poorly understood • Induce structural changes? Interfere with receptor interactions? • Prevent uncoating by aggregation? Bivalent cross-linking? • Different hosts use different mechanisms depending on the virus • Antibody-mediated response to infectious entity must work in tandem with other defense mechanisms (opsonization)

22. Human RhinoVirus-14

23. HRV14 • Cryo Stuctures for HRV-14 complexed with Fab fragments 17-IA and 12-IA from stongly neutralizing but weakly aggregating Mab • Also complexed with Fab fragment of weakly neutralizing but strongly aggregating Mab • Entire IgG (Mab 17-IA) • Fab/Mab fully saturates HRV14 virion