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Background:  Astroviruses   are   a   major   cause   of   diarrhea   in  c h i l d r e n   a n d   immuno comp rom i s e d  popula5ons.   Infec5ons   are   seasonal   in   nature,  following  the  rainy  season  in  tropical  areas  and  the   winter   season   in   temperate   areas.   The  different   serotypes   have   varying   clinical  presenta5ons,   with   diarrhea,   fever,   and  vomi5ng  being  common  symptoms.  

Human   astroviruses   (HAstV)   are   non-­‐enveloped,  single-­‐stranded,  posi5ve  sense  RNA  viruses.   The   HAstV   capsid   protein   contains   5  dis5nct   structural   domains.   One   of   these  (known  as  the  acidic  domain),  is  thought  to  be  involved  in  virus  matura5on  and  assembly.  

                   

Methods:  • Cloning   was   performed   to   produce   a  pET52b   expression   plasmid   containing   the  acidic  domain  nucleo5de  sequence  

• Protein   was   expressed   in   the   BL21   E.   coli  cell  line  

• Purifica9on   was   performed   by   affinity   and  size  exclusion  chromatography  

• Computa5onal   tools   were   used   to   predict  secondary  and  ter5ary  structure  

• Nuclear   magne5c   resonance   (NMR)   and  circular   dichroism   (CD)   were   performed   to  corroborate   predicted   secondary   structural  characteris5cs  

• A   trypsin   digest   was   performed   to   isolate  the  puta5ve  helical  bundle  

• Screening   trays   were   set   up   to   find   ideal  crystalliza9on  condi5ons

Acknowledgments:    !

! ! Thank you to Professor DuBois for acting as my senior thesis advisor, and to Crown College !! ! for project funding.

Results:  Cloning,   expression,   and   purifica5on   produced   an  ample  supply  of  stable  acidic  domain  protein.    

Conclusions:  While   the   ter5ary   structure   of   the   acidic  domain   is   s5ll   unknown,   protocols   for  successfully   producing   and   purifying   it   have  been  demonstrated  here.  

Several   lines   of   evidence   (including   tryp5c  digest   paWern,   circular   dichroism,   and   sizing  behavior)   have   helped   confirm   the   predicted  secondary  structure  of  approximately  40%  helix  and  60%  unstructured.  

AWempts   to   crystallize  both   the  en5re  domain  and   the   puta5ve   helices   isolated   by   tryp5c  digest  were  ul5mately  unsuccessful.  

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!

Elizabeth  A.  LagesseDuBois Lab, University  of  California,  Santa  Cruz

Structural Studies of the Human Astrovirus 1 Capsid Acidic Domain

SDS  PAGE  aHer  size  exclusion  chromatography  showing  the  acidic  domain  protein  as  a  bright  band  just  below  25  kDa

Computa5onal   tools   predicted   an   N-­‐terminal  disordered   region   and   a   C-­‐terminal   region  consis5ng  of   four  helices.  Several   ter5ary  structure  predic5ons  were  also  produced.  

RobeLa  secondary  structure  predic9on,  showing  predicted  helices  as  red  “H”s

NMR   and   CD   studies   were   consistent   with   a  secondary   structure   of   approximately   40%   helices  and  60%  unstructured  regions.

2  of  5  RoseLa  ter9ary  structural  predic9ons

To   isolate   the   helices,   limited   proteolysis   was  performed   with   trypsin.   The   result   was   a   stable  band   present   at   all   protease   concentra5ons.   This  band  was  separated  using  a  size  exclusion  column.

Full   length   protein   and   trypsinized   fragment   were  concentrated   and   used   to   prepare   crystalliza5on  condi5on   screening   trays.   Unfortunately,  degrada5on   of   the   fragment   had   already   occurred  due  to  residual  trypsin  in  the  sample.    

No  crystals  were  observed   in   full   length  protein  or  intact   fragment.     A   variety   of   condi5ons  produced  crystals   in   the   degraded   fragment,   but   even   these  were  bundles  of  crystals,  unsuitable  for  diffrac5on.  

Trypsin  digest  concentra9on  test  ranging  from  10:1  to  2500:1  acidic  domain:trypsin  ra9o  (by  mass).  The  red  arrow  corresponds  to  the  full  length  construct,  and  the  blue  arrow  corresponds  to  the  stable  band

 Crystal  bundles  formed  from  degraded  protein  sample

Domain  diagram  for  the  human  astrovirus  capsid  protein

Future  Direc9ons:  !

•   Cloning   was   performed   to   produce   an  expression   plasmid   with   only   the   helical  regions   of   the   acidic   domain.   (avoiding  t ryps in   d iges5on)   Express ion   and  purifica5on   tests   will   be   performed   using  this  new  construct.  

!•   Func5onal   assays   are   currently   in  development,   which   aim   to   localize   the  protein  within  the  cell  and  determine  what  effect   (if   any)   this   domain   has   on  mammalian   cells   without   the   presence   of  the  full  virus.  !•   If   crystalliza5on   proves   difficult,   a  structure  for  one  or  both  constructs  may  be  pursued  via  NMR.

Electron  micrographs  of  a  single  immature  astrovirus  (leH)  and  a  group  of  mature  astroviruses  (right).  (Dreyden  et.  al.  2012,  Crea9ve  Commons  2.0,  respec9vely)