Viral Assembly through Host
DefenseKristin ShinglerJuly 28, 2011
Modulation of T-Number
P2 P4
P4ProCapsid P4
gpSid
T7 vs T4 ConformationP4 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
HepB 7.4- and 9-A Reconstructions
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
Assembly of Big VirusesQuasi-Equivalence is plausible for small T
numbers, but less so as the number increases
Example: Algal Virus Paramecium bursaria Chlorella Virus Type 1 T=169 1900-A diameter
Large complex viruses utilize scaffold and accessory proteins to aid in assembly
Adenoviruses
Difference ImagingIdentified 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
Herpes Simplex Virus-1 T=16
Procapsid ComponentsVP5 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.
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
Cowpea Chlorotic Mottle Virus
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
Flock House Virus
FHVX-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
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
Alphavirus Budding
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.
Virus TransmissionLittle 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
Antibody Structure
Host DefenseVertebrate 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)
Human RhinoVirus-14
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