Viral Assembly through Host Defense

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