ad5 poster final.pptx

Simultaneous In situ detection of adenovirus 5 genomic DNA and RNAs in human cells culture and tissue Krzywkowski  Tomasz1,  Ci1ci  Sibel2,  Punga  Tanel2  &  Nilsson  Mats1     1  Department  of  Biochemistry  and  Biophysics,  Stockholm  University,  Science  for  Life  Laboratory,  Stockholm,  Sweden     2  Department  of  Medical  Biochemistry  and  Microbiology,  Uppsala  University,  Uppsala,  Sweden  

Introduc)on  and  background   Adenoviruses   (Ad)   can   infect   variety   of   cell   types   and   hosts   worldwide.   SymptomaIc   infecIons   most   commonly   lead   to   respiratory   tracts   diseases.   While   being   rarely   fatal   in   humans,   Ad   infecIons   consItute   serious   threat   in   organ   transplant   recipients   with   deficient   immune  response.  Most  frequent  ly)c  model  of  Ad5  infecIon  results  in  cell  lysis  and  release  of  new  parIcles  within  48h.  During  latent   infecIon  in  B-­‐  and  T-­‐cell  populaIons,  virus  is  not  eliminated,  but  persists  in  infected  Issues  and  can  reacIvate  in  immunocompromised   paIents.   Understanding   mechanisms   underlying   Ad5   infec)on   and   persistency   down   to   the   single   cell   level   is   important   yet   challenging  with  available  techniques.    

Method   Novel  method  comprises  series  of  enzymaIc  steps,  including  targeted  RNA  reverse  transcripIon,  endo-­‐/exo-­‐nucleolyIc  ad5  DNA  fragmentaIon,   followed   by   molecular   profiling   with   padlock   probes   and   Rolling   Circle   AmplificaIon   (RCA).   RCA   products   are   fluorescently   "decorated",   facilitaIng  quanIficaIon  of  individual  targets  in  automated  fashion  in  situ.  DetecIon  of  RNA  (E1A  –  "early"  viral  RNAs;  MLTU  –  "late"  viral  RNAs)   and  virus  genomic  DNA  combined  with  RCA  enables  accurate  molecular  evaluaIon  of  infected  cells  with  great  specificity  and  sensiIvity.  

Results  -­‐  E1A  early  RNAs  are  accumulaIng  throughout  lyIc  infecIon      

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6hpi                                                                                13hpi                                                                              18hpi                                                                                24hpi    

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Figure  2.  In  situ  detec)on  of  ad5  genomic  DNA  and  virus  associated    RNAs  during  ly)c  infec)on  in  human  cells.   HeLa   cells   were   infected   and   fixed   ager   0-­‐24   hours   post   infecIon   as   indicated.   White-­‐   ACTB,   green   –   E1A,   red   –   MLTU,   magenta   –   ad5   genomic  DNA.  Scale  bar  :  20μm.  Increasing  nuclear  accumula=on  is  evident  on  the  boAom  figures  where  viral  DNA  is  not  shown.   E1A$cytoplasm$

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Plot  1.  Quan)ta)on  of  average  signal/cell  for  ad5  genomic  DNA   and  RNAs  during  ly)c  infec)on  in  human  cells.   Significant  increase  in  signal  for  all  targets  but  ACTB  is  observed   ager   6hpi.   Nuclear   accumulaIon   of   E1A   is   shown   while   cytoplasmaIc  presence  of  E1A  is  stable.    

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Plot   2.   E1A   expression   ra)o   between   cell   nucleus/cytoplasm   during  the  ly)c  infec)on  and  in  E1A  transfected  cells.   In  late  infecIon  stages,  E1A  nucleus/cytoplasm  raIo  is  very  high   (~24).   The   accumulaIon   was   not   observed   in   E1A   transfected   cells  (~0.4).  

Latent  infecIon  in  human  lymphocytes;  method  validaIon  in  clinical  samples   Figure  3.  In  situ   detec)on  of  ad5   genomic  DNA  and   virus  associated     RNAs  during   persistent  infec)on  in   BJAB  cells.   Human  lymphocytes   were  infected  with   ad5  and  fixed  ager  6   days.     Scale  bar  20  μm  

A                                                                          B                                                                                        C   Figure  4.  In  situ  detec)on  of  ad5  genomic  DNA  and  virus  associated     D   RNAs  in  infected  tonsil.  

X  and  Y  coordinates  for  all  signals  are  ploced  back  on  H&E  stained   clinical  specimen.  A)  SuperimposiIon  of  all  signals,  scale  bar  50  μm;  B)   E1A  and  MLTU;  C)  ad5  genomic  DNA  and  ACTB;  D)  magnified  area  is   depicted  on  the  Issue  scan  with  a  red  square.  

Figure  1.  Simplified  detec)on  schema)cs  of  ad5  DNA  and  mRNAs.     Ad5   genomic   DNA   (green)   is   sequenIally   cleaved   with   endonuclease   and   exonuclease  to  generate  ssDNA  strand  (red).  mRNAs  of  interest  are  converted  to   cDNA(blue)   with   targeted   reverse   transcripIon.   DegradaIon   of   RNA   renders   cDNA   single   stranded.   Introduced   padlock   probes   (violet)   specifically   recognize   DNA/RNA   targets   and   become   circularized   with   DNA   Ligase.   Ligated   padlock   probe   and   exposed   3'   hydroxyl   group   of   DNA   (arrows)   serve   as   a   substrate   to   iniIate  RCA.  Products  of  amplificaIon,  long  tandemic  copies  of  original  padlock   probe   collapse   into   ~500nm   "balls"   that   are   fluorescently   labeled   with   short   fluorophore-­‐conjugated  complementary  oligonucleoIdes.    

Discussion   We  present  herein  a  novel  method  for  simultaneous   in   situ   detecIon   of   ad5   DNA   and   RNA   down   to   the   single   cell   level   in   various   cell   cultures   and   paIents   tonsils.   We   observe   that   RNAs   transcribed   from   adenovirus   early   region   1A   (E1A)   unit   are   gradually   accumulaIng   in   cells   nuclei   throughout   24h   lyIc   infecIon  in  HeLa  cells  (see  Plot  1  and  2).  In  contrast,   expression   of   RNAs   associated   with   late   infecIon   stages   (MLTU)   exhibit   both   nuclear   and   cytoplasmic   localizaIon.   We   show   that   our   method   provides   a   valuable   tool   to   study   ad5   lyIc/latent   infecIon   mechanisms   and   heterogeneity   in   both   cell   cultures   and  clinical  samples.   Authors  contributed  equally  to  this  work*