Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1 Christina R. Bourne, M. G. Finn, and Adam Zlotnick University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, and The Scripps Research Institute, La Jolla California Contact 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. References • 450 million people have chronic HBV infection − that’s more than HIV or Hepatitis C • Leading cause of liver disease and liver cancer • 1 million deaths per year [1]. • Small enveloped doubled stranded DNA virus • Icosahedral shape (20 triangular faces) • Four protein chains, A-D, arranged as AB and DC dimers (identical moelcules that are linked together) What is Hepatitis B Virus? To determine structure, HBV capsid’s were crystallized with and without HAP1 bound. How to crystallize a structure? Significance Results •Identification of the HAP1-binding site is promising corresponds to structural changes for activation and capsid assembly misdirection •Capsid structure seen as an early stage in capsid disruption process due to HAP1’s capsid- destabilizing activity • Effects of HAP1 on assembled capsids and the assembly process observed by the fivefold vertices protusion from capsid surface threefold vertices increased opening quasi-sixfold vertices flattening Capsid slightly expands overall Discussion Although a vaccine exists, knowledge of the structural basis for HAP1 activity can be used for developing capsid-targeting anti-viral strategies. Further investigations into the HAP1 binding site is a great target for stopping viral production. Conclusions Methods and Materials Sources Why HAP1 molecule? Zlotnick, Mukhopadhyay (2011) Science Direct, 19:14-23 Previous studies show HAP1 significantly decreased virus production by: 1. increasing rate of capsid formation 2. causing misshaped particles as concentration increased Purpose of research: How does HAP1 specifically change HBV’s capsid structure? Where does HAP1 bind on the HBV coat? •Determining the structural basis for HAP1 activity can be used for developing capsid-targeting anti- viral strategies. •Currently most anti-HBV therapies target enzymes with the downfall that there is human analog to every viral enzyme •Targeting viral capsid proteins is very favorable since there are no human homologs. Comparison of capsid structure with and without HAP1 Overlay BLUE – HAP1 RED +HAP1 Figure 4. Dimers shown as AB+CD [2]. Figure 2. Dimer arrangement with and without HAP1 Normal HBV capsids Misshaped HBV capsids at higher HAP1 amounts Stephen et. al. (2005) PNAS 102(23):8138-8143 Capsid (virus shell made of proteins) 1. Threefold vertices increase opening in size 2. The AB dimer pivots upward causing the fivefold vertices protrude from capsid surface - HAP1 +HAP1 3. Quasi-sixfold vertices flatten 4. Likely binding sites for HAP1 identified (red) Vertices of Icosahedral symmetry 5fold 3fold 2fold/quasi-6fold Overlay: grey –HAP1, color +HAP1 X-rays are shot through the crystal create a diffraction pattern which is used to produce an electron density map that is used to determine the structure of the molecule. This process is repeated many times and the average is taken for an accurate structure [4]. 1.Zlotnick, mukhopadhyay, (2011) Science Direct, 19;14-23 2.Schinazi RF (2014) Drug Discovery Towards HBV Golbal Eradication. Slideshow presentation. Paris June, 17, 2014. 3.Stephen et al. (2006) PNAS 102(23):8138-8143 4.Fendler, Bernard, and Brad Groveman. "X-Ray Crystallography and It's Applications." Lecture. Florida State University Program in Neuroscience. Web. 21 Sept. 2010. <www.neuro.fsu.edu/~dfadool/BFendler.ppt> Figure 2. Dimer arrangement of quasi-sixfold and threefold vertices with presumptive HAP1 binding positions on the end of the C chains Contact: Kristin Cox Email: [email protected]

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Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1

Christina R. Bourne, M. G. Finn, and Adam Zlotnick University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, and The Scripps Research Institute, La Jolla California

Contact1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

References

• 450 million people have chronic HBV infection− that’s more than HIV or Hepatitis C

• Leading cause of liver disease and liver cancer• 1 million deaths per year [1].

• Small enveloped doubled stranded DNA virus

• Icosahedral shape (20 triangular faces)• Four protein chains, A-D, arranged as AB and DC dimers (identical

moelcules that are linked together)

What is Hepatitis B Virus?

To determine structure, HBV capsid’s were crystallized with and without HAP1 bound. How to crystallize a structure?

Significance

Results •Identification of the HAP1-binding site is promising corresponds to structural changes for activation and capsid assembly misdirection

•Capsid structure seen as an early stage in capsid disruption process due to HAP1’s capsid-destabilizing activity

• Effects of HAP1 on assembled capsids and the assembly process observed by

the fivefold vertices protusion from capsid surface threefold vertices increased openingquasi-sixfold vertices flatteningCapsid slightly expands overall

Discussion

Although a vaccine exists, knowledge of the structural basis for HAP1 activity can be used for developing capsid-targeting anti-viral strategies.

Further investigations into the HAP1 binding site is a great target for stopping viral production.

Conclusions

Methods and Materials

Sources

Why HAP1 molecule?

Zlotnick, Mukhopadhyay (2011) Science Direct, 19:14-23

Previous studies show HAP1 significantly decreased virus production by: 1. increasing rate of capsid formation 2. causing misshaped particles as concentration increased

Purpose of research: How does HAP1 specifically change HBV’s capsid structure? Where does HAP1 bind on the HBV coat?

•Determining the structural basis for HAP1 activity can be used for developing capsid-targeting anti-viral strategies.

•Currently most anti-HBV therapies target enzymes with the downfall that there is human analog to every viral enzyme

•Targeting viral capsid proteins is very favorable since there are no human homologs.

Comparison of capsid structure with and without HAP1

OverlayBLUE – HAP1RED +HAP1

Figure 4. Dimers shown as AB+CD

[2].

Figure 2. Dimer arrangement with and without HAP1Normal HBV capsids Misshaped HBV capsids at higher HAP1 amounts

Stephen et. al. (2005) PNAS 102(23):8138-8143

Capsid (virus shell made of proteins)

1. Threefold vertices increase opening in size

2. The AB dimer pivots upward causing the fivefold vertices protrude from capsid surface

- HAP1 +HAP1

3. Quasi-sixfold vertices flatten 4. Likely binding sites for HAP1 identified (red)

Vertices of Icosahedral symmetry

5fold 3fold 2fold/quasi-6fold

Overlay: grey –HAP1, color +HAP1

X-rays are shot through the crystal create a diffraction pattern which is used to produce an electron density map that is used to determine the structure of the molecule. This process is repeated many times and the average is taken for an accurate structure [4].

1. Zlotnick, mukhopadhyay, (2011) Science Direct, 19;14-232. Schinazi RF (2014) Drug Discovery Towards HBV Golbal Eradication.

Slideshow presentation. Paris June, 17, 2014.3. Stephen et al. (2006) PNAS 102(23):8138-81434. Fendler, Bernard, and Brad Groveman. "X-Ray Crystallography and It's

Applications." Lecture. Florida State University Program in Neuroscience. Web. 21 Sept. 2010. <www.neuro.fsu.edu/~dfadool/BFendler.ppt>

Figure 2. Dimer arrangement of quasi-sixfold and threefold vertices with presumptive HAP1 binding positions on the end of the C chains

Contact: Kristin CoxEmail: [email protected]