Yotam Hulata 12.09.14

Course 138001: Sex, life and development

 
 
 
Do    Archaeal    viruses    have    different    release    strategies    depending    on  
their  morphology?  
 
Abstract
 
Background:   Living   organisms   are   divided   into   three   separated   domains:   Eukarya,  
Bacteria   and   Archaea.   Although   Archaea   look   similar   to   Bacteria   in   their   structure,  
their   gene   expression   mechanisms   have  many   similarities  to   Eukarya.   Archaea   can   be  
found   in   the   most   extreme   environments   and   at   moderate   environments   as   well.  
Wherever   life   exists,   viruses   are   found.   In   this   context,   Archaeal   viruses   are   very  
interesting.   The   first   archaeal   viruses   discovered   belong   to   the   Myoviridae   and  
Siphoviridae  families  in  the  order  of  head-­‐tail  viruses.  In  recent  years,  various  unique  
virus   morphtypes   have   been   discovered   among   archaeal   viruses:   fusiform,   bottle-­‐  
shaped,   droplet-­‐shaped,   linear   and   spherical.   A   virus   has   to   attach   to   the   cell   and  
enter   its   genome   into   it   and   finally   to   be   released   from  the   cell   after   reproduction.  
The   post   infection   release   strategies   of   the   head-­‐tail   phages   are   well   described   for  
bacterial   viruses.   Recently,   a   unique   releasing   strategy,   of   a   rod-­‐shaped   archaeal  
virus,  has  been  described.  
 
Research   plan:   My   hypothesis   is   that   viruses   with   different   morphologies   have  
different   release   strategies.   To   test   this   I   will   examine   the   infection   cycle   of   two  
archaeal   viruses   with   different   morphologies.   Infected   cells   from   the   time   points   in  
which  viruses  are  released  will  be  examined  by  electron  microscopy.  
 
Significance:   This   research   will   give   us   a   better   understanding   about   the   infection  
cycle  of  archaeal  viruses  focusing  on  their  releases  strategies.  
 
 
 
 
Subject and specific aims of the proposal
 
Back   in   the   70’s,   when   Carl   Woese   looked   at   the   ribosomal   RNA   and   ribosomes  
sequences,   he   found   out   that   the   world   of   living   organisms   actually   divide   to   three  
separate   domains   and   not   to   two   as   thought   before   when   classified   based   only   on  
the   cell-­‐ultrastructure.   These   domains   are   Bacteria,   Eukarya   and   Archaea   (Woese  
and   Fox   1977).   Since   those   days   archaea   have   been   extensively   studied   and   today  
our   knowledge   about   them   is   much   wider   (Garrett   et   al   2006).   One   of   the   insights  
discovered   about   archaea   is   their   capability   to   live   at   very   extreme   and   moderate  
environments.   In   many   aspects,   mostly   in   their   structure,   archaea   resemble   to  
bacteria,   but   when   looking   on   DNA   replication,   transcription   and   translation   they  
show   similarity   to   eukarya.   Still   there   are   some   features   that   are   unique   to   the  
archaea  (i.e.  ether-­‐linked  membranes)  (Prangishvili  et  al  2006).  

 
   Yotam Hulata 12.09.14

 
Course 138001: Sex, life and development
 
 
Viruses  are  the  most  abundant  life  form  on   earth.  Wherever  life  exists,  viruses  have  
been   found   (Suttle   2007).   The   first   archaeal   viruses   isolated   belonged   to   the   head-­‐  
tail   order.   In   the   last   ten   years,   electron-­‐microscopy   images   from   habitats   that  
contain  mostly  archaea  revealed  that  actually  the  head-­‐tail  viruses  are  rare.  Instead,  
those   works   show   exceptional   morphotypes   of   the   archaeal   viruses.   Among   those  
morphotypes   we  can  find  the  bottle  shaped  viruses,   droplet  shaped  viruses,   fusiform  
viruses,   linear   viruses,   spherical   viruses   and,   as   mentioned   earlier,   the   head-­‐tail  
viruses     (Prangishvili     et     al     2006).     Viral     life     cycles     contain     a     few     major     steps:  
attachment   to   the   host,  expression   of   its  genetic  material,   assembly  of   the   virion   and  
release  of  the  new  virions  from  the  cell.  
 
The  release  strategies  of  the  new  virions  from  the  head-­‐tail  order  are  well  studied  in  
the   bacteria.   There   are   two   distinct   strategies   for   releasing   from   the   cell.  
Bacteriophages  that  contain  large  genomes  use  a  holin-­‐endolysin  system  (Wang  et  al  
2000),  while  small  genome  phages  use  a  single  lysis  protein   (Bernhardt  et  al  2002).  
Moreover,  it  has  been  shown  that  the  λ-­‐  phage  and  the  φX174  phage  that  belong  to  
the   Siphoviridae   and   Myoroviridae   families   respectively   and   that   have   different  
morphologies   also   have   different   release   strategies.   The   λ-­‐   phage   needs   three   lysis  
genes,   while   the   φX174   phage   has   only   one   necessary   and   sufficient   gene   for   lysis  
(Altman   et   al   1985).   Recently,   a   unique   release   strategy   has   been   described   in  
archaeal   virus.   In   their   work,   Bize   et   al   (2009),   showed   that   a   rod-­‐shaped   archaeal  
virus  created  at  10  hours  after  infection  pyramidal  protrusions  over  the  cell  surface.  
At  13  hours  after  infection,  those  pyramidal  structures  were  absent  and  instead  the  
cell  surface  contained  holes.  At  26  hours  the  cell   contain  mostly  holes  and  it   lost  its  
natural  morphology  (Bize  et  al  2009).  
 
As  mentioned  above,  archaea  viruses  have  a  variety  of  morphologies  but  in  contrast  
to   the   bacteria   viruses,   a   comparison   between   the   virus   morphology   to   its   release  
strategy  has  not  been  done.  
 
My  specific  aims  are:  
 
1.       Characterize     the     life     cycle     of     two     different     archaeal     viruses     that     have  
different   morphology   but   have   a   similarity   in   other   properties   (e.g.   genomic  
material).  
2.       Learn   the   infection   kinetic   of   these   viruses   with   emphasis   on   the   release  
period.  
3.       Characterize  the  release  strategy  of  those  two  viruses  by  two  methods.  
a.       EM  images  of  infected  archaea  culture.  
b. Genome  sequencing  of  the  archaeal  viruses  and  protein  expression.  

 
   Yotam Hulata 12.09.14

Course 138001: Sex, life and development

 
 
Significance of the proposed research
 
Viruses   are   major   players   in   control   of   the   archaea   population.   Understanding   the  
release   strategies   of   the   different   morphologies   of   archaeal   viruses   will   give   us   a  
better   understanding   on   the   viruses’   life   cycle   and   about   the   competing   of   the  
archaea  with  their  viruses.  
 
My   hypothesis   is   that   different   morphologies   in   archaeal   viruses   have   different  
releasing   strategy.   If   this   hypothesis   will   be   proven,   it   will   mean   that   releasing  
methods  could  be  predicted  based  on  the  virus  morphology.  
 
 
 
 
Experimental design and methodology
 
Host   strain   and   viruses   isolation:   The   hyperthermophilic   Acidianus   genus   will   be  
grown   at   75oC,   pH   3.   50   ml   of   culture   will   be   incubated   with   1   ml   of   water   sample  
from   the   crater   of   the   Solfatara   volcano   at   Pozzuoli,   Italy,   to   isolate   the   dsDNA  
Acidianus   bottle-­‐shaped   virus   (ABV)   (Haring   et   al   2005).       The   hyperthermophilic  
Sulfolobus   genus   will   be   grown   as   the   above   and     the   dsDNA   Sulfolobus  
neozealandicus   droplet   virus   (SNDV)   will   be   isolated   from   Icelandic   Solfataras   (Zillig  
et  al  1993).  
 
In  order  to  verify  which  viruses   I  have  I  will  perform  a  PCR  procedure  and  amplified  
the     specific     genes     from     these     two     viruses.     The     amplicon     will     be     cloned     and  
sequenced  for  verification.  
 
Characterization   of   the   infection   cycle:       in   order   to   figure   out   what   are   the   times  
after   infection   in   which   the   new   virions   start   to   be   released   from   the   cell   I   will   use  
the   One-­‐step   procedure.   The   two   model   archaea   for   this   work   –   Acidianus   and  
Sulfolobus   neozealandicus   will   be   infected   with   their   viruses   –   ABV   and   SNDV  
respectively.   After   infection,   samples   will   be   taken   for   plaque   assay   in   several   time  
points   after   infection.   I   expect   to   see,   at   the   beginning   of   the   experiment,   a   high  
numbers   of   viruses   and  then   reduction   in   their  numbers   to   a  minimum.  This  period  
of   time   after   infection   is   the   time   in   which   the   viruses   adsorbed   to   the   cells.   After  
this,  I  expect  to  see  a  rise  in  the  amount  of  viruses  to  a  maximum.  This  period  is  the  
time  in  which  the  new  virions  start  to  release  from  the  cells.   By  using  the  One-­‐step  
procedure   I   can   learn   about  the  kinetic   of   the   infection   cycle,   the   length   of   the   latent  
period,   the   length   of   the   lytic   period   and   also  the   burst   size.   The   differences   in   the  
infection   kinetic   in   general   and   in   the   lytic   period   and   burst   size   in   particular   can  
imply  on  different  release  strategies.  

 
   Yotam Hulata 12.09.14

 
Course 138001: Sex, life and development
 
 
Characterization   of   the   release   strategy:   in   order   to   reveal   the   different   release  
strategies   I   will   take   three   samples   of   infected   culture.   The   first   time   point   will   be  
when   the   new   virions   start   to   release.   The   second   time   point   that   will   be   taken   is  
from   the   time   in   which   the   release   is   in   the   middle   of   the   process.   The   third   time  
point  is  when  new  virion   is  released.  The   data  for   the  time   points  will  come  from  the  
One-­‐step   procedure   above.   All   the   samples   will   be   observed   with   transmission  
electron   microscopy   (TEM)   and  scanning   electron   microscopy   (SEM)   (Bize   et   al   2009).  
By   using   ultrathin   sections   TEM   images   I   would   be   able   to   notice   in   changes   that  
occur   the   cell   from   the   inner   side   of   the   cell   while   using   SEM   will   give   me   a   better  
look   on   the   outside   of   the   cell.   The   EM   images   will   allow   me   to   compere   between  
the  two  releases  strategies  of  the  different  viruses.  
 
Another  approach  that  I  will  use  in  order  to  look  for  different  release  strategies  will  
be   to   sequence   the   genomes   of   both   of   the   viruses   and   look   for   known   homolog  
genes  related  to  release.   Comparing  between  the   genes  that  related  to   the  releasing  
of   the   new   virions   will   give   a   batter   understanding   about   their   release   strategy.  
Based   on   the   sequencing   I   will   do   a   proteome   analysis   at   the   release   time   point   in  
order  to  check  which  proteins  are  present  during  the  release  period.  
 
 
 
 
References
 
Altman  E,  Young  K,  Garrett  J,  Altman  R,  Young  R  (1985).  Subcellular  Localization  of  Lethal  
Lysis  Proteins  of  Bacteriophages  λ  and  φX174.  Journal  of  Virology  53:  1008-­‐1011.  
Bernhardt  TG,  Wang  I-­‐N,  Struck  DK,  Young  R  (2002).  Breaking  free:  "protein  antibiotics"  and  
phage  lysis.  Research  in  microbiology  153:  493-­‐501.  
Bize  A,  Karlsson  EA,  Ekefjärd  K,  Quax  TEF,  Pina  M,  Prevost  M-­‐C  et  al  (2009).  A  unique  virus  
release  mechanism  in  the  Archaea.  PNAS  106:  11306-­‐11311.  
Garrett  R,  Klenk  H-­‐P,  (eds)  (2006).  Archaea.  Evolution,  Physiology  and  Molecular  Biology.  
Blackwell  Publishing:  Oxford.  
Haring  M,  Rachel  R,  Peng  X,  Garrett  RA,  Prangishvili  D  (2005).  Viral  Diversity  in  Hot  Springs  of  
Pozzuoli,  Italy,  and  Characterization  of  a  Unique  Archaeal  Virus,  Acidianus  Bottle-­‐  
Shaped  Virus,  from  a  New  Family,  the  Ampullaviridae.  Journal  of  Virology  79:  9904-­‐  
9911.  
Prangishvili  D,  Forterre  P,  Garrett  RA  (2006).  Viruses  of  the  Archaea:  a  unifying  view.  
NATURE  REVIEWS    MICROBIOLOGY  4:  837-­‐848.  
Suttle  CA  (2007).  Marine  viruses  —  major  players  in  the  global  ecosystem.  Nature  Reviews  
Microbiology  5:  801-­‐812.  
Wang  I-­‐N,  Smith  DL,  Young  R  (2000).  Holins:  The  protein  clocks  of  bacteriophage  infections.  
Annu  Rev  Microbiol  54:  799-­‐825.  
Woese  CR,  Fox  GE  (1977).  Phylogenetic  structure  of  the  prokaryotic  domain:  The  primary  
kingdoms.  Proc  Natl  Acad  Sci  USA  74:  5088-­‐5090.  

 
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Course 138001: Sex, life and development
 
 
Zillig W, Kletzin A, Schleper C, Holz I,Janekovic D, Hain Jet a/ (1993). Screening for
Sulfolobales, their Plasmids and their Viruses in Icelandic Solfataras. Systematic and
Applied Microbiology 16: 609-628.