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Figure  5:  Structures  of  the  HIV-­‐1  genome  and  virion.  A)  The  HIV-­‐1  genome  is  composed  of  two  flanking   LTRs   containing   typcial   retroviral   regulatory   sequences,   and   different   ORFs   coding   for   gag-­‐pol,   env,   the   regulatory  proteins  rev  and  tat,  and  the  accessory  proteins  vif,  vpr,  vpu  and  nef.  The  rev-­‐responsive  element   (RRE,  light  blue  box)  is  depicted  below  the  env  ORF  (green  box).  B)  The  structure  of  the  HIV-­‐1  mature  virion   is  depicted  with  the  cleaved  viral  products,  which  are  indicated  with  arrows.  A)  and  B)  from  Sakuma  et  al,   2012  80.  

 

1.1.8  The  HIV-­‐1  life  cycle    

 

 The  first  step  of  the  HIV-­‐1  replication  cycle  is  the  viral  particle  fusion  with  the   plasma  membrane  of  a  susceptible  cell  that  expresses  specific  receptors.  Upon   the  binding  of  gp120  to  the  CD4  receptor  of  an  immune  cell  such  as  a  

lymphocyte,  it  further  interacts  with  a  specific  seven  transmembrane  domain  G   protein-­‐coupled  coreceptor  depending  on  the  virus  tropism.  Generally,  CCR5-­‐

tropic  virus  dominate  the  first  phase  of  the  infection,  and  gradually  the  tropism   of  some  strains  can  change  to  reach  up  to  50%  of  viral  particles  that  use  the   alternative  CXCR4  co-­‐receptor  81,82.  Additionally,  some  strains  of  HIV-­‐1  are  able   to  use  other  coreceptors  as  CCR2b,  CCR3,  CCR8  and  the  orphan  receptors  V28,   STRL33  and  GPR15  83.  

 

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 Once   the   virus   fuses   with   the   membrane,   the   viral   core   is   released   into   the   cytoplasm,  where  reverse  transcription  takes  place.    It  is  still  a  matter  of  debate   where   the   capsid   uncoating   may   occur,   before   reverse-­‐transcription,   concomitantly   or   after   completion   of   the   process.   Three   major   models   are   postulated,  as  reviewed  in  84.  One  of  them  put  forward  the  hypothesis  that  the   total  uncoating  is  necessary  to  activate  the  reverse-­‐transcription  complex  (RTC).  

This   first   hypothesis   was   based   on   the   observations   that   low-­‐levels   of   capsids   were  recovered  from  HIV-­‐1  complexes  extracted  from  cells  and  on  the  inability   to  visualize  the  capsid  complexes  with  Transmission  Electron  Microscopy  (TEM)   within  infected  cells  85-­‐87.  These  findings  were  not  able  to  discriminate  between   partial  and  total  uncoating.  

 

 Based  on  the  results  from  a  study  showing  that  CA  associates  with  the  RTC,  two   other  schools  of  thoughts  emerged  88.  The  first  alternative  model  implies  that  the   capsid   uncoating   happens   gradually,   upon   environmental   changes   encountered   by   the   viral   core,   like   the   interaction   with   different   cellular   factors   at   different   steps   of   the   reverse-­‐transcription   process   and   the   production   of   cDNA   and   reverse-­‐transcription  intermediates.  This  hypothesis  is  reinforced  from  one  side,   by  the  observation  of  different  sizes  of  HIV-­‐1  cores  isolated  from  early  infected   cells  and  that  diverge  from  the  mature  HIV-­‐1  complexes  88,89.  From  another  side,   the   findings   that   altering   the   stability   of   the   capsid   core   via   mutagenesis   of   specific  CA  residues  to  either  an  increase  or  a  decrease  will  affect  the  completion   of   the   reverse-­‐transcription   are   in   agreement   with   this   model  90.   Whereas   the   first  observation  could  simply  represent  an  artifact  coming  from  the  biochemical   method  used  for  the  virus  isolation,  the  second  finding  uses  mutations  that  could   induce   conformational   changes   that   modify   the   interactions   with   host   factors   and  the  RTC  in  in  vivo  experiments  that  are  not  relevant  in  the  course  of  a  natural   infection.   Nevertheless,   TRIM5,   a   factor   that   will   be   described   later,   blocks   retroviruses  at  an  early  infection  step,  via  accelerated  CA  core  disassembly.  This   observation   reinforces   the   model   in   which   a   tightly   controlled   uncoating   is   necessary  for  an  efficient  viral  replication.  

 

 The  second  alternative  model  postulates  that  the  capsid  remains  associated  with   the   RTC   until   completion   of   the   reverse-­‐transcription   at   the   nuclear   pore.   The   finding  that  the  capsid  is  required  for  proper  nuclear  import  84,91  argues  in  favor   of  this  model,  but  does  not  contradict  the  postulate  where  uncoating  is  a  gradual   event.  

Alternatively,   the   uncoating   could   possibly   not   occur,   as   shown   by   the   experiments  done  by  Burdick  and  colleagues  92.  

 

 Nevertheless,  the  CA  uncoating  of  at  least  a  portion  of  particles  seems  to  happen   very   early   after   viral   entry,   as   shown   by   experiments   using   the   CA-­‐specific   blockade   of   HIV-­‐1   by   TRIM5Cyp,   which   will   be   described   later.   TRIM5Cyp-­‐

mediated  restriction  is  mediated  by  recognition  of  intact  cores  and  this  binding   is   impeded   by   Cyclosporine   A   (CsA),   as   refered   below.   Taking   out   the   CsA   treatment   (CsA   washout   assay)   after   2   hours   precludes   definitively   restriction,   indicating  that  the  CA  stability  was  disrupted  before  this  time  point  93.  Similarly,   immunofluorescence  microscopy  allowed  detecting  less  particles  associated  with   capsid  after  1  hour.  In  the  same  study,  the  chemical  inhibition  of  the  RT,  using   nevirapine  (NVP),  in  combination  with  the  CsA  washout  assay  showed  that  the   reverse-­‐transcription   progression   is   necessary   for   normal   uncoating,   as   this   process   did   not   become   apparent   when   the   inbitory   drug   was   used  93.   The   restriction  factor  TRIM5,  recognizes  the  capsid  hexameric  lattice.  It  is  therefore   possible  that  even  if  only  partial  uncoating  happen,  TRIM5Cyp  wouldn’t  bind  to   the  particle  anymore.  Further  evidence  that  uncoating  is  a  rapidly  started  event   comes   from   a   study   where   another   set   of   restriction   factors,   the   APOBEC3   proteins,  act  on  the  nascent  cDNAs,  in  the  target  cell  94.  This  finding  suggests  that   the  RTC  is  accessible  very  early  within  the  viral  core  to  proteins  present  in  the   cytoplasm,  before  or  at  the  time  of  reverse-­‐transcription  95.  

 

 Concomitantly,  there  are  several  results  suggesting  that  CA  remains  associated   until  a  late  step  of  retroviral  replication.  First,  experiments  show  that  CA  is  the   determinant   for   nuclear   import  71,96,97.   Second,   CA   interacts   with   Cyclophilin   A   (CypA)   and   this   interaction   is   required   for   proper   reverse-­‐transcription   but   blocks  nuclear-­‐entry  in  some  cell  lines  98.  Third,  a  study  revealed  that  CA  binds  to  

the  CypA  domain  of  the  Nucleoporin  protein  of  358  kDa  (Nup358)  at  the  nuclear   pore   complex.   Interaction   with   this   protein   allows   nuclear   entry   of   the   PIC  99.   When   Nup358   or   another   member   of   the   complex   involved   in   this   nuclear   internalization   pathway   -­‐   TNPO3   or   cytoplasmic   CypA-­‐   are   disrupted,   other   routes   are   used   and   result   in   different   preferential   integration   sites   and   concomitant   impaired   HIV-­‐1   replication,   as   reviewed   by   Fassati  95.   Fourth,   CA   binds  to  nuclear  export  factors,  suggesting  it  can  localize  to  the  nucleus  100.  Fifth,   CA   total   uncoating   is   not   necessary   for   reverse-­‐transcription   to   proceed,   as   a   mutant  that  stabilizes  the  CA  core  still  synthesizes  normal  levels  of  cDNA  93.    

 The  observation  that  different  natural  or  artificial  restriction  factors  recognizing   the  CA  block  different  pre-­‐integration  steps,  including  post-­‐nuclear  entry,  further   argues   in   favor   of   a   partial   and   gradual   uncoating   that   culminates   into   the   nucleus  at  some  step  before  integration  101,102  103.  Despite  a  considerable  body  of   work  devoted  to  the  HIV-­‐1  uncoating  process,  the  subcellular  compartment  and   the  viral  replication  step  where  its  completion  occurs  remain  a  mystery.  What  is   clear  from  previous  studies  is  that  a  proper  timing  of  CA  uncoating  is  necessary   for  various  steps  of  the  viral  life  cycle  to  proceed,  as  shown  by  the  CA-­‐dependent   negative   effect   on   HIV-­‐1   replication   by   restriction   factors   that   accelerate   the   uncoating  (see  below)  and  by  proteins  that  are  influencing  nuclear  entry.  

 

 The  process  of  the  nuclear  import  of  the  PIC  seems  to  rely  on  the  capsid  protein.  

In   earlier   studies,   IN,   MA   and   Vpr   were   suggested   to   be   required   for   nuclear   entry   and   the   fact   that   these   proteins   carry   a   nuclear   localization   signal   (NLS)   supported  this  theory.  To  understand  the  key  experiments  that  were  performed   to  investigate  the  viral  proteins  involved  in  nuclear  import  of  the  PIC,  one  must   consider   that   there   is   a   major   difference   between   lentiviruses   and   other   retroviruses  like  the  gammaretrovirus  MLV,  in  respect  to  nuclear  entry.  

As   such,   whereas   HIV-­‐1   can   enter   the   nucleus   of   non-­‐dividing   cells,   MLV   is   dependent  on  the  breakdown  of  the  nuclear  membrane  at  the  mitosis  to  import   its   PIC  104-­‐106.   This   feature   in   fact   allows   the   gammaretrovirus   to   uncoat   after   nuclear  entry  107,  perhaps  conferring  a  protection  of  the  RTC  and  the  PIC  from   cytoplasmic   sensors.   Taking   advantage   of   this   difference,   the   IN   or   the   CA  

proteins  of  both  retroviruses  were  exchanged,  expecting  that  they  could  confer  a   differential  ability  to  enter  the  nucleus.  

 

 When   adding   a   NLS   into   the   MLV   MA   or   IN   proteins,   MLV   could   still   not   be   imported   into   the   nucleus   of   resting   cells  108,109.   Interestingly,   however,   the   exchange   of   the   MLV   capsid   by   the   one   from   HIV-­‐1,   transferred   to   the   gammaretrovirus   the   independency   from   cell-­‐cycle   requirements   for   nuclear   entry  91.   Reciprocally,   HIV-­‐1   with   an   MLV   capsid   lost   its   ability   to   infect   non-­‐

dividing  cells.    

 

 To   import   the   PIC   into   the   nucleus,   nuclear   pore   complex   (NPC)   proteins   as   Nup98,  Nup153  and  Nup358  and  other  cellular  factors  seems  to  be  required,  as   reviewed  by  Fassati  95.  Before  nuclear  entry,  the  PIC  machinery  already  activates   the   viral   DNA   to   be   integrated  69.   IN   binds   to   both   viral   LTRs,   recognizing   internal   specific   sequences   and   catalyzes   the   processing   at   a   CA   dinucleotide,   producing   an   available   3’   hydroxyl   group,   which   constitute   a   cleaved   donor   complex  (CDC)  that  is  competent  for  nucleophilic  attack  of  the  target  DNA  once   in  the  nucleus.  This  interaction  leads  to  the  DNA  strand  transfer  in  whom  the  3’  

ends  of  the  viral  cDNA  are  ligated  to  5’  phosphates  into  the  host  chromatin,  as   reviewed   by   Krishnan   and   Engelman  69.   Once   the   strand   transfer   complex   is   formed,   repair   enzymes   from   the   host   take   care   of   joining   and   filling   the   gaps   created  at  the  5’  ends  of  viral  cDNA  within  the  targeted  host  genome,  creating  the   so  called  target  site  duplication  (TSD)  at  both  ends  of  the  provirus  69.  

 

 As   reviewed   by   Karn   and   Stolzfus  78,   regulation   of   HIV-­‐1   expression   from   the   provirus  is  controlled  both  transcriptionally  and  post-­‐transcriptionally.  The  two   accessory   proteins   tat   and   rev   are   responsible   for   the   stimulation   of   the   transcripts  elongation  and  the  export  of  some  mRNAs  species,  respectively,  that   would   otherwise   be   degraded   within   the   nucleus.   Within   HIV-­‐1   LTR,   the   transactivation-­‐responsive  element  (TAR)  recruits  tat  and  its  cellular  cofactor  P-­‐

TEFb,   resulting   in   transcriptional   elongation,   a   process   that   is   dependent   on   cellular  elongation  factors  as  ELL2  78.  

 

 The  core  promoter  of  HIV-­‐1  contains  three  SP1  binding  sites,  a  TATA  box  and  an   initiator   sequence.   Additionally,   the   HIV-­‐1   LTR   bears   an   NFKB   binding   site,   which   acts   a   viral   enhancer   involved   in   the   reactivation   of   latency   and   in   increased  HIV-­‐1  replication  in  T  cells  78,110.  The  epigenetic  regulation,  dependent   on  acetylation  and  methylation  of  histones  as  well  as  on  DNA  methylation  allows   the  virus  to  establish  latency  111.  At  a  later  stage,  HIV-­‐1  transcripts  are  exported   from  the  nucleus  to  the  cytoplasm.  Given  that  unspliced  and  incompletely  spliced   mRNAs  are  the  target  of  nuclear  enzymes  that  degrade  them,  these  transcripts   species  have  to  associate  with  rev,  that  recognize  a  specific  sequence  in  the  env   coding   sequence,   the   rev-­‐responsive   element   (RRE)   and   hide   them   from   the   cellular  machinery.  The  protected  transcripts  are  then  exported  through  the  NPC   via  interaction  with  the  cellular  protein  Crm1  78.  

HIV-­‐1  transcripts  are  further  processed  at  the  3’  end  and  polyadenylated,  in  the   view  of  being  translated  together  with  host-­‐derived  mRNAs  78.  

 

 The  gag   gene   products   that   constitute   the   structural   components   of   the   HIV-­‐1   virion   coordinate   the   last   phase   of   viral   replication.   Indeed,   viral   proteins   and   nucleic   acid   materials   assembly   at   the   viral   membrane   is   directed   by   the   unprocessed   gag   polyprotein   that   binds   the   plasma   membrane,   and   the   env   protein,  recruits  the  PR,  the  RT  and  the  IN  proteins  and  packs  the  viral  RNA  and   the   primer   tRNALys2,   3  112,   forming   an   immature   virion.   During   the   budding   process,  the  plasma  membrane  is  integrated  into  the  viral  particle,  constituting  a   de   novo   lipid   bilayer.   Upon   particle   maturation,   the   PR   protein   cleaves   gag,   producing   processed   MA,   CA   and   NC.   The   resulting   mature   virions   are   either   released   into   the   blood   stream   or   directly   infect   new   cells   via   cell-­‐to-­‐cell   transmission  involving  the  formation  of  virological  synapses  39,113.  

 

 My  thesis  will  focus  on  the  activity  of  the  restriction  factor  TRIM5,  which  will  be   introduced  further  in  the  next  chapter,  and  thus  I  will  examine  the  early  steps  of   retroviral  replication.  

   

1.2  TRIM5  and  the  innate  immunity    

 Viruses   and   other   pathogens   attack   the   organism   by   different   routes.   The   immune   response   that   is   mounted   to   counteract   this   invasion   depends   on   different  germline-­‐encoded  and  de  novo  synthesized  factors  that  are  produced  in   the  context  of  the  innate  and  adaptive  immune  response,  respectively.    As  a  first   defense,   all   cell   types   that   are   the   target   of   infections   carry   different   combinations   of   proteins   acting   like   sentinels   that   recognize   specific   foreign   motifs   or   molecular   signatures.   These   pathogen-­‐associated   molecular   patterns   (PAMPs)   are   bound   by   pattern-­‐recognition   receptors   (PRRs),   at   the   cell   membrane,   in   endosomal   compartments   or   within   the   cytoplasm,   and   this   interaction   results   in   the   activation   of   signaling   pathways   that   will   ultimately   lead  to  the  production  of  inflammatory  cytokines  and  type  I  Interferon  (Type  I   IFN),   as   reviewed   in  114The   inflammatory   cytokines   activate   immune   cells   and   act  on  endothelial  cells,  in  this  way  stimulating  the  early  inflammatory  response   (reviewed  in  115).  

 

 Secretion   of   Type   I   IFN   provokes   an   antiviral   state   via   the   production   of   interferon-­‐stimulated  genes  (ISGs)  and  the  stimulation  of  the  acquired  immune   system   by   activating   immune   cells,   contributing   to   the   presentation   of   Major   Histocompatibility  Complex  Class  I  (MHC  I)  molecules,  essential  for  recognition   of  antigens  exposed  by  antigen-­‐presenting  cells  (APCs)  and  promoting  cytotoxic   T  lymphocytes  (CTL)  response  116.  

 

 Type   I   IFN   stimulates   the   expression   of   a   plethora   of   factors   with   different   specificities  to  mount  a  broad  antiviral  response.  The  category  of  ISGs  contains   several   hundreds   of   genes   that   serve   as   antiviral   effectors   or   as   signaling   activators   for   processes   such   as   apoptosis   and   vesicular   transport  117.   Many   antiviral   effectors   are   components   of   the   intrinsic   immunity,   and   will   be   discussed   later.   These   constituvely-­‐expressed   factors   are   able   to   degrade   viral   components  specifically  in  a  direct  way.  

Interestingly,  as  it  will  be  discussed  later,  some  of  these  ISGs,  including  TRIM5,   function  as  PRR  them  selves,  showing  the  way  by  which  the  innate  immunity  can   be  self-­‐amplified.  

 

1.2.1  The  Pattern-­‐recognition  receptors    

 

 Four   main   families   of   PRR   are   associated   with   detection   of   foreign   material   within  a  cell:  one  group  of  membrane-­‐associated  receptors  and  three  classes  of   cytoplasmic  PRRs.  

Toll-­‐like  receptors    

 

 A  first  group  of  PRRs  is  composed  of  proteins  with  a  transmembrane  domain,  an   extracellular   leucine   rich   repeat   (LRR)   region   that   recognizes   specific   PAMPs,   and   an   intracellular   module   containing   a   TLR/IL-­‐1R   (TIR)   domain   that   allows   them  to  interact  with  adaptors  molecules  for  the  signal  transduction  118.  

 

 With   ten   functional   members   in   humans,   the   Toll-­‐like   receptor   (TLR)   family   recognizes   a   wide   variety   of   PAMPs.   Using   different   adaptor   molecules,   TLRs   bind   to   many   types   of   molecules   including   lipids,   lipoproteins,   proteins   and   nucleic  acids.  The  members  of  this  family  of  receptors  are  located  at  different  cell   compartments.  Whereas  TLRs  1,  2,  4,  5,  6  and  10  are  found  at  the  cell  surface,   TLRs  3,  7,  8,  and  9  are  located  in  endosomes  114,119  (figure  6).  TLR2  is  found  in   the  form  of  a  dimer,  either  with  TLR1,  TLR6  or  TLR10  mainly  detecting  PAMPs   from   Bacteria   and   fungi   114,120,121.   When   complexed   with   TLR1,   the   dimer   recognizes   the   triacetylated   lipoproteins,   peptidoglycans   and   lipopolysaccharides  122,  as  reviewed  by  Kawai  and  Akira  114.  

The  dimer  composed  of  TLR2  and  6  is  responsible  for  the  detection  of  diacylated   lipoproteins  123.   The   specific   molecules   recognized   by   TLR2-­‐TLR10   have   not   been  discovered  yet  120,121.  The  other  cell  surface-­‐associated  PRRs,  TLR4  and  5,   were   found   to   bind   to   lipopolysaccharides   (LPS)   and   flagellin  124,   respectively     (reviewed  in  125).