Structural Virology

Question 1

Viral Crystallography

Answer 1

• solves structures of viral proteins
• atomic resolution important for target development and optimisation of selectivity/potency
• can determine structure of virus particles
• viruses with rigid protein shell (no lipid envelope)

Question 2

NMR

Answer 2

• restricted to smaller proteins
• useful for proteins that won’t crystallise
• useful for studying interactions and dynamics between proteins
  eg. shift mapping of VPg interactions with eIF4g (controls translation start)

Question 3

Cryo EM

Answer 3

• used for larger structures both symmetric and nonsymmetric
• solves structure to atomic resolution
  eg. Hepatitis C Virus complexes with 40s ribosomal subunit

Question 4

Cryo Electron Tomography

Answer 4

• imaging particles not symmetric or homogenous enough for crystallisation or single particle analysis
• track infection events
• image proteins inside particles

Question 5

FMDV

Answer 5

• foot and mouth disease
• picornavirus: single strand positive sense RNA genome
• single ORF able to self cleave into viral proteins
• VP: viral proteins are 4 capsid proteins forming a complex encapsulating new genomic RNA

Question 6

3C Protease

Answer 6

• cleaves 10 junctions in precursor peptide
• allows virus to assemble in host
• cuts itself out within same chain or acts on others

Question 7

Antiviral Target

Answer 7

• 3C is a good target because it is mostly conserved among serotypes
• drug targeting this will affect other serotypes
• is not a surface protein and is highly conserved due to key function
• VP tolerate mutation for increased fitness from immune system

Question 8

Structural Determination of 3C Protease

Answer 8

• WT insoluble when expressed in E Coli
• mutated surface residues to increase solubility without affecting structure/function
• mutated C142 to S for increased solubility and crystals

Question 9

3C Structure

Answer 9

• active site in cleft between 2 beta barrels
• flexible protease (auto-proteolysis)
• B ribbon (no disordered loop)

Question 10

Catalytic Triad of 3C

Answer 10

• Cys/His/Asp
• similar mechanism to chymotrypsin: His abstracts proton and Cys conducts nucleophilic attack while Asp stabilises system

Question 11

Hepatitis A Virus

Answer 11

• thought to be a catalytic dyad of Cys and His
• crystallisation artefact: packing interactions distorted active site
• was in fact as Asp

Question 12

Problems with crystals

Answer 12

• substrate binding site obscured by crystal packing
• not able to soak candidate compounds
• designed mutants in surface residues of original crystal contacts to disrupt contacts

Question 13

Enzyme Activity

Answer 13

• C142S mutation reduced activity
• Serine is similar in structure but activity was horrid
• wild type is active but prone to aggregation
• mutation of C142 to hydrophobic residues greatly increased activity

Question 14

Cleavage Specificity

Answer 14

• specificity determinants spread of range of sub-sites
• recognition of amino acids either side of bond
• viral proteases very specific (between p4 and p2’)

Question 15

FMD Substrate Binding

Answer 15

• binding cleft expands to accomodate substrate
• L47 flips position to create a pocket for leucine in P1 position to bind to protease
• pocket created by movement of leucine out of way to create interaction
• H bond interactions over 7 amino acids

Question 16

C142 Problem

Answer 16

• tip of B ribbon structure
• forming hydrophobic contacts with side chain
• postioned in between side chain in P2/P4
• proline and lysine (long methylene chain)
• hydrophobicity of this position positions subtrate accurately in active site
• serine causes misalignment such that scissile bond not presented well to active site

Question 17

FMD Vaccine

Answer 17

• used ability to create mutants with dialed down protease
• need chemically inactive virus
• requires growing of large tissue cultures infected with virus and then purified
• significant risks
• new method: used fragment of genome experssing 3 main capsid proteins and 3C protease
• mini polyprotein cleaved and assembles into capsids
• without genome the capsids are non infectious
• overexpression of protease is toxic (reduce 3C levels while keeping capsid levels)
  1. frameshift mutation so that 3C is not always made in equimolar ratios
  2. incorporated one mutation discovered to reduce 3V activity
• allows overexpression of synthetic viral particles
• also enhance stability of emtpy capsule with engineered disulfide bond

Question 18

Coronavirus

Answer 18

• trimeric surface spike glycoprotein
• lipid enveloped
• positive sense single stranded monopartite RNA genome
• RNA coated in nucleoprotein (helical look)

Question 19

SARS1 Genome

Answer 19

• 5’ cap and 3’ polyA tail
• many ORF
• ORF1a-b translated as single polyprotein
• may terminate at end of ORFa or have a framshift for continuation
• pp1a and pp1ab polyproteins cut into component nsps by two viral proteases
• spike protein gene
• envelope protein gene
• accessory proteins

Question 20

CoV Proteome

Answer 20

• nsp5: main 3C like protease
• nsp7+8: interact to form hexadecameric ring
• nsp12: RNA dependent RNA pol
• S: spike
• N: nucleocapsid

Question 21

SARS CoV Replication

Answer 21

1. binding and entry of host
2. translation of positive snse RNA to a replicase
3. transcription to negative small genomic RNAs for protein product and replication to form positive sense genomic RNA
4. assembly into nucleocapsid
5. forms endoplasmic reticulum-golgi intermediate compartment
6. release from host

Question 22

Nucleoprotein Structure

Answer 22

• N terminal RNA binding domain
• C terminal dimerization domain
• potential flexible middle domain

Question 23

Spike Structure

Answer 23

• homotrimer
• each mono cleaved into S1 and S2 fragments that are noncovalently associated
• S1 contains the receptor binding domain and S2 has the fusion peptide
• 2 states: close and partially open (one RBD flipped up)
• recognition of receptor drives S2 hydrophobic peptide into target membrane to trigger fusion (conformational change)

Question 24

Spike Protein Fusion

Answer 24

• only in open state is the RBD accessible to ACE2 on host cell
• proteolytic processing of S protein at surface releases fusion peptide for insertion into host membrane to initiate fusion

Question 25

Fusion Mechanism

Answer 25

1. when S protein is translated the subunits are covalentyl bonded 2. S1/S2 site cleaved and noncovalent bonding 3. RBD binds to ACE2 in up conformation hooking virus to cell surface 4. S protein cleaved again at S2' site to activate fusion 5. major conformational changes leading to membrane fusion 6. delivery of genome to host cell

Question 26

nsp7+8 structure

Answer 26

- from polyprotein pp1a - hexadecameric complex - potential processivity factor for RdRp

Question 27

Protease structure

Answer 27

- nsp5 is main 3C like protease | - catalytic dyad of Cys and His

Question 28

Drug Fragment Screening Project

Answer 28

- soak protein in drug fragments - bound compounds solved to see binding patterns - try to develop fragments that bind closely and distinctly into novel drugs

Question 29

RNA dependent RNA polymerase

Answer 29

- nsp12 - part of polyprotein pp1ab - partial double stranded RNA template is inserted into the central channel of RdRp - drug candidate Remdesivir is covalently incorporated into primer strand at first replicated base pair and terminates chain elongation

Question 30

Benefits of Structural Understanding of Covid

Answer 30

- structural understanding can help refine constructs - structure of spike protein helps design of constructs to make primary antigen - can help us understand evolution of spike protein to develop strongest immune response