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1.2.3  Immunity  to  retroviruses:  restriction  factors    

 

 The  replication  ability  of  retroviruses  in  different  cells  depends  on  many  cellular   factors.   The   first   considered   factor   is   the   entry   of   the   retrovirus   into   the   cell   cytoplasm,   via   recognition   of   the   corresponding   receptor.   For   example,   as   discussed  previously,  HIV-­‐1  entry  requires  the  recognition  of  the  CD4  receptor   and  a  coreceptor,  principally  CXCR4  or  CCR5.  The  subsequent  steps  of  the  viral   life   cycle   exploit   host   proteins   in   a   species-­‐dependent   way   to   proceed,   as   highlighted  by  the  inability  of  HIV-­‐1  to  productively  infect  murine  cell  lines  that   have  been  engineered  to  express  human  CD4  182  and  taking  into  account  that  the   murine   CXCR4   can   be   used   as   a   coreceptor   by   HIV-­‐1  183.   Importantly,   murine   cells  have  a  cyclin  T1  protein,  that  HIV-­‐1  Tat  does  not  bind  because  of  a  species-­‐

specific   polymorphism,   thus   precluding   the   employment   of   this   cofactor   required   for   the   transactivation   of   LTR-­‐directed   expression   184.   When   circumventing   this   post-­‐entry   blocks   by   expression   of   human   Cyclin   T1,   some   murine   cell   lines   proceed   into   viral   gene   transcription,   but   further   steps   are   blocked,  as  mRNA  export  and  processing,  as  well  as  virion  assembly  185,186.  These   blockades   are   rescued   upon   fusion   of   murine   and   human   cells,   showing   that   there  are  factors  exerting  a  positive  effect  on  viral  replication  late  steps  that  are   not  present  in  the  mouse  187.  

 

   Interestingly,   in   contrast   to   fibroblasts,   murine   T   cells   do   not   support   HIV-­‐1   reverse   transcription  183.   The   blockade   of   a   pre-­‐integration   step   of   the   viral   replication   strongly   recalls   other   phenotypes   observed   in   mice   and   primates.  

The  cell  tropism  is  not  only  dictated  by  the  presence  or  the  absence  of  positive   cofactors  in  a  cell.  

   

The   first   indirect   report   of   a   negative   factor   influencing   retroviral   replication   was   in   1957   by   C.   Friend   who   discovered   that   a   genetic   transmissible   trait  

dictated   the   susceptibility   of   different   strains   of   mice   to   MLV  188.   The   factor   mediating   this   blockade   was   later   genetically   mapped   on   chromosome   4   and   called  Fv-­‐1  189,190.  The  two  alleles  of  the  gene  were  Fv1B  and  Fv1N  that  conferred   the  resistance  to  N  tropic  MLV  (N-­‐MLV)  and  B-­‐MLV,  respectively.  Nineteen  years   after   Friend’s   discovery,   the   blockade   by   Fv-­‐1   was   determined   to   act   after   reverse-­‐transcription,  but  before  integration  191  (figure  8).  

 

 In  parallel  with  the  previous  findings,  a  potent  blockade  of  HIV-­‐1  infection  was   observed  in  monkey  cells  192,193  and  was  termed  lentiviral  susceptibility  factor  1   (Lv1).   Few   years   later,   an   Fv-­‐1B-­‐like   restriction   was   observed   in   cells   from   primates  including  human,  and  from  dog,  pig  and  cow  that  potently  blocked  N-­‐

MLV   early   post-­‐entry  194.   The   host   protein   responsible   for   this   blockade   was   called  restriction  factor  1  (Ref1).  

 

 In   common   to   all   restriction   factors,   the   barrier   to   retroviruses   was   capsid-­‐

specific,  dominant  and  saturable  by  a  high  amount  of  viral  particles  195-­‐199.  The   factors  at  the  origin  of  these  blockades  were  all  cloned  and  it  appeared  that  Lv1   and  Ref1  were  products  of  the  same  gene,  TRIM5  62,200-­‐203.  

 

 I  will  first  briefly  describe  the  best-­‐studied  restriction  factors  affecting  retroviral   replication  and  will  then  focus  on  TRIM5.  

 

 

Figure  8:  The  blockade  of  early  retroviral  replication  steps  by  TRIM5  and  Fv1.  The  restriction  factors   TRIM5   and   Fv1   inhibit   retroviral   replication   at   an   early   post-­‐entry   step.   Whereas   TRIM5   can   act   before   reverse-­‐transcription  and  nuclear  import  (solid  black  bars),  Fv1  only  targets  the  latter  step.  The  virion  core   is  represented  by  the  blue  conical  shape.  RNA  and  DNA  species  within  the  core  are  depicted  as  two  black   and  blue  bars,  respectively.  Courtesy  of  Prof.  Jeremy  Luban  (adapted).  

 

APOBEC3  proteins    

 Among   a   family   of   cytidine   deaminases,   the   apolipoprotein   B   mRNA-­‐editing   enzyme   catalytic   polypeptide-­‐like   3G   (APOBEC3G)   inhibits   the   replication   of   HIV-­‐1   by   associating   with   assembling   virions,   via   its   N   terminal   zinc-­‐binding     deaminase  domain  that  interacts  with  the  viral  RNA  and  gag  polyprotein  204,205.   Once   in   a   target   cell,   APOBEC3G   recognizes   cytosine   residues   within   C-­‐C   dinucleotides  on  newly  synthesized  minus-­‐strand  viral  cDNA  and  induces  their   deamination,   transforming   it   into   a   uracil   206.   The   resulting   viral   genome   contains  guanine  to  adenine  mutations,  leading  to  replication  catastrophe  207.  In   addition,  the  catalytic  activity  of  APOBEC3G  is  required  for  the  blockade  of  HIV-­‐1   integration.  The  mechanism  relies  on  the  interference  with  the  tRNALys3  primer   dissociation,  leading  to  the  formation  of  abnormal  3’LTRs  and  thus  a  subsequent   defect  in  its  targeting  to  the  host  genome  208.  Finally,  APOBEC3G  targets  HIV-­‐1  at  

TRIM5 Reverse Transcription

Nuclear Import Fv1

the  reverse-­‐transcription  step  by  impeding  the  tRNALys3  to  prime  the  viral  RNA,   although   it   is   not   clear   whether   this   is   in   a   deaminase-­‐dependent   way  209-­‐212.   However,  despite  these  potent  restrictions,  HIV-­‐1  evolved  a  mean  to  counteract   APOBEC3G  by  orchestrating  its  degradation  by  the  viral  accessory  protein  Vif  in   a  proteasome-­‐dependent  pathway  206.  

 

 Other   members   of   the   APOBEC   family   have   similar   deamination   activity   and   restrict  HIV-­‐1  infection  213.  While  APOBEC3A  has  been  linked  to  the  inhibition  of   HIV-­‐1   in   monocytes  214,   APOBEC3B   is   not   expressed   in   primary   lymphoid   cells   but   still   renders   HIV-­‐1   particles   less   infectious,   when   expressed   transiently   in   the  virus-­‐producing  cells  213,215.  The  other  APOBEC  proteins  exerting  anti-­‐HIV-­‐1   activity  include  APOBEC3C  that  inhibits  the  infectivity  of  some  strains  of  HIV-­‐1  

216,  APOBEC3D/E  that  is  counteracted  by  vif  217  and  APOBEC3F  213,218,219.      

Tetherin    

 The   tetherin   restriction   factor,   named   in   that   way   because   it   “tethers”   HIV-­‐1   virions  to  the  cell  surface,  impeding  their  release  220.  By  homodimerizing  via  the   extracellular   coiled-­‐coil   domain,   tetherin   engage   a   second   monomer   bound   to   the  viral  membrane  221.  

 

 Tetherin  is  induced  by  type  I  IFN  and  its  action  is  counteracted  by  HIV-­‐1  Vpu  220.   In  turn,  the  activation  of  the  NFκB  pathway  by  tetherin  upon  HIV-­‐1  infection  222   results  in  the  production  of  type  I  IFN.  

 

SAMHD1    

At   first   associated   with   the   Aicardi-­‐Goutières   autoimmunity   syndrome  223   the   sterile   alpha   motif   (SAM)   and   histidine-­‐aspartic   (HD)   domains-­‐containing   protein  1  (SAMHD1)  was  subsequently  investigated  for  its  role  in  mediating  the   innate   immunity   to   retroviruses.   This   restriction   factor   was   found   to   decrease   dNTP  levels  and  to  block  HIV-­‐1  reverse-­‐transcription  224.  As  for  APOBEC3G  and   tetherin,   some   retroviral   accessory   proteins   neutralize   SAMHD1.   Indeed,   Vpx  

from  HIV-­‐2  and  SIV  degrades  SAMHD1  by  targeting  this  factor  to  the  proteasome  

225.     MX2    

The  IFN-­‐induced  myxovirus  resistance  2  (MX2)  protein  restrict  HIV-­‐1  infection   in  a  capsid-­‐dependent  way  103.  Although  the  mechanism  of  retroviral  inhibition   remains  unknown,  the  transient  expression  of  the  restriction  factor  decreased  2-­‐

LTR  circles  formation  and  integration  103,  suggesting  that  this  protein  inhibits   HIV-­‐1  nuclear  entry.  

    ZAP    

The   first   retroviral   target   of   the   zinc-­‐finger   antiviral   protein   (ZAP)   to   be   discovered   was   MLV  226.   In   this   study,   the   abundance   of   MLV   transcripts   was   decreased  in  rodent  cells  expressing  the  endogenous  protein.  It  was  later  found   that  HIV-­‐1  was  similarly  restricted  by  the  human  ZAP  orthologue,  which  induced   specific  mRNA  uncapping  and  degradation  of  the  retroviral  transcripts  227.  

 

MOV10    

 Discovered   in   murine   strains   as   the   site   of   MLV   provirus   integration  2.   the   MOV10   gene   encodes   a   protein   with   seven   helicase   motives  228.   The   human   orthologue   of   MOV10   inhibits   HIV-­‐1   at   various   replication   steps.   Although   the   mechanism  by  which  MOV10  reduces  HIV-­‐1  virion  production  is  still  unclear,  it   could  involve  the  inhibition  of  gag  expression  229  and  this  could  have  a  link  with   the   observed   association   of   MOV10   orthologues   from   mammals   with   the   RNA   interference   (RNAi)   system  230   that   may   silence   viral   gene   expression.     At   a   second   level,   virion-­‐associated   MOV10   from   the   producer   cells   restricts   HIV-­‐1   reverse-­‐transcription  in  the  target  cells  229,231.  

 

ADAR-­‐1    

 The   adenosine   deaminase   acting   on   RNA   protein   1   (ADAR1)   induces   the   deamination  of  adenosine  into  inosine  on  a  double-­‐stranded  RNA  substrate  232.  It   was  recently  found  that  this  enzyme  has  a  restriction  activity  on  HIV-­‐1,  inhibiting   the   expression   of   viral   proteins   via   the   post-­‐transcriptional   mRNA   editing   inducing  a  defect  on  nuclear  export  of  the  respective  messengers  of  gag,  pol  and   env  233.  

  Fv1    

 The   sequence   of   the   murine   restriction   factor   Fv1   is   derived   from   a   gag   gene   from   the   endogenous   retrovirus   family   ERV-­‐L   present   across   mammalian   genomes,  as  revealed  by  the  approximated  60%  of  homology  with  the  sequence   of   human   ERV-­‐L   (HERV-­‐L)  234.   The   resulting   capsid-­‐like   protein   recognizes   specific  capsids  of  MLV  strains.  The  different  alleles  of  Fv1,  N  and  B,  differ  only  in   three  residues  within  a  small  motif  associated  with  their  restriction  capacity  235   and  recognize  differentially  a  residue  at  position  110  of  the  amino  acid  sequence   of  the  CA  protein  of  B-­‐  and  N-­‐MLV,  respectively  236.  For  the  binding  between  the   MLV  capsid  and  Fv1  to  happen,  the  gag  polyprotein  must  be  mature  and  cleaved   from  p12  and  NC  237.  The  direct  binding  was  shown  using  a  biochemical  method   where   capsid-­‐coated   lipid   nanotubes   were   subjected   to   immunoprecipitation   with  Fv1  proteins.  As  observed  by  negative  staining  and  electron  microscopy,  the   capsid  units  assembled  in  an  ordered  manner  in  vitro,  dependent  on  the  typical   retroviral  β-­‐sheet  formation  on  the  N  terminus  of  the  CA  protein  238.  Importantly,   the  binding  results  were  in  agreement  with  the  specific  restriction  pattern  of  the   distinct  Fv1  alleles  238.  

  TRIM5    

 The  large  family  of  TRIpartite  Motif  (TRIM)  proteins  comprises  approximately   100   members   in   human   239.   Besides   a   few   exceptions,   they   all   share   the   conservation   of   three   main   modules   in   a   precise   order   (reviewed   in  240).   The   Ring-­‐finger  (RF)  domain  is  found  at  the  N-­‐terminal  part  of  a  TRIM  protein.  This  

domain   is   formed   of   zinc-­‐coordinating   motifs,   that   allows   the   formation   of   a  

“cross-­‐brace”   structure,   involving   cysteins   and   a   histidine   that   contact   two   Zn   atoms  241-­‐243.  

Present  in  several  families  of  proteins,  RF  domains  often  confer  binding  to  an  E2-­‐

ubiquitin  conjugating  enzyme  and  function  as  E3-­‐ubiquitin  ligases  to  themselves   and  to  other  substrates,  as  reviewed  by  Deshaies  and  Joazeiro  243.  Indeed,  many   TRIM   family   members   have   been   described   to   display   E3-­‐ubiquitin   ligase   activity,   including   TRIM5,   TRIM21   and   TRIM25  244-­‐246,   as   reviewed   in  240.   The   nuclear  magnetic  resonance  (NMR)  structure  of  the  human  TRIM5α  RING  finger   has  been  solved  247  and  showed  that  the  core  of  the  domain  is  composed  of  the   majority  of  the  hydrophobic  residues  and  is  located  between  two  β-­‐sheets  and   an  α-­‐helix.  

 

 As  a  second  motif,  TRIM  proteins  carry  one  or  two  B-­‐box  domains,  which  share  a   similar   ternary   conformation.   Indeed,   B-­‐boxes   also   coordinate   Zn   atoms   in   a   crossed  configuration  and  form  two  β-­‐sheets,  followed  by  a  α  helix,  as  shown  by   the  study  of  MID1  (TRIM18)  and  human  TRIM5α  248-­‐250.  The  TRIM  B-­‐box1  always   precedes  B-­‐box2  and  is  never  found  alone  240.  In  contrast,  many  TRIMs  possess   only  the  B-­‐box2,  as  exemplified  by  TRIM5  240.  The  function  of  B-­‐box  domains  is   not   completely   understood.   However,   the   study   of   the   B-­‐box2   of   MuRF1   (TRIM62)  revealed  a  surface  hydrophobic  patch  with  polar  residues  on  a  dimer   interface  251.  This  finding  allowed  the  manipulation  of  the  equivalent  residues  of   TRIM5   B-­‐box2   and   showed   that   this   domain   is   important   for   protein   higher-­‐

order  multimerization  250,252.      

 As  a  third  motif,  the  Coiled-­‐coil  (CC)  domain  is  the  last  module  of  the  tripartite   RING   finger-­‐B-­‐box-­‐CC   (RBCC)   motif.   It   contains   appropriately   spaced   hydrophobic   residues   on   amphipathic   α-­‐helices,   forming   two   putative   leucine-­‐

zipper  motives,  which  are  responsible  for  TRIM  protein-­‐to-­‐protein  interactions  

253,254.   This   domain   was   shown   to   participate   in   the   higher-­‐order   multimerization,  homo-­‐  and  hetero-­‐dimer  formation  of  TRIM  proteins  as  well  as   to  be  required  for  their  concentration  into  discrete  cellular  compartments  such   as  the  nuclear  and  cytoplasmic  bodies  (NB  and  CB,  respectively)  255-­‐259.  

 

 The  TRIM  proteins  vary  in  their  C-­‐terminal  domain  composition.  As  reviewed  by   Ozato  and  colleagues  240,  there  are  ten  distinct  C-­‐terminal  TRIM  domains,  found   alone  or  in  different  combinations,  and  participating  to  functions  as  variable  as   localization   to   microtubules,   binding   to   a   retroviral   capsid   or   interaction   with   histones  and  transcriptional  repression.  The  discovery  that  the  TRIM5  gene  was   the  determinant  for  HIV-­‐1  restriction  in  monkey  cells  62,200,201,203,  motivated  the   study  of  the  involvement  of  TRIM  proteins  in  the  innate  immunity.  Importantly,   TRIM25  was  found  to  induce  the  K63  polyubiquitination  RIG-­‐I,  essential  for  the   RNA   viruses-­‐triggered   signal   transduction  245.   Although   TRIM1,   TRIM19   and   TRIM22  were  already  shown  to  have  antiviral  properties  (as  reviewed  by  Nisole   and  colleagues  260),  a  large  screen,  looking  at  55  TRIM  proteins  revealed  that  this   feature   was   shared   by   many   other   TRIM   family   members  261.   Notably,   TRIM11   and  TRIM15  were  found  to  restrict  the  release  of  HIV-­‐1  and  MLV,  respectively,   with   TRIM15   recognizing   the   gag   protein   in   the   producer   cell   in   a   B-­‐box-­‐

dependent  way  261.    

 The  role  of  TRIM  proteins  in  the  innate  immune  response  is  further  emphasized   by   the   fact   that   their   expression   is   up-­‐regulated   upon   type   I   IFN   treatment   or   induction  of  TLR  by  agonists  and  that  they  differentially  activate  the  AP-­‐1,  NFκB   and  IFNβ  promoters  262-­‐266.  

 

1.2.4  TRIM5-­‐mediated  retroviral  restriction    

 Six  isoforms  have  been  described  for  the  human  TRIM5  gene,  namely  α,  γ,  δ,  ε,  ι   and   κ   267.   Only   TRIM5α   have   been   shown   to   inhibit   retroviral   replication.  

Moreover,   TRIM5ι   is   the   second   more   abundant   transcript   in   some   human   cell   lines  and,  similar  to  the  isoforms  γ,  δ  and  κ,  down-­‐regulates  TRIM5α  levels  and   correspondingly  modulates  the  anti  N-­‐MLV  restriction  activity  267.  An  important   particularity   of   TRIM5α   is   that   it   is   the   only   isoform   that   possess   a   C-­‐terminal   PRY-­‐SPRY  domain  (figure  9).  

 

   

Figure  9:  Schematic  representation  of  the  TRIM5  orthologues  and  structure  of  the  proteins.  TRIM5   proteins  are  composed  of  an  RBCC  motif,  comprising  the  RING  finger  (RF),  the  B-­‐Box  (BB)  and  the  Coiled-­‐

Coil   (CC).   Some   TRIM5   orthologues   carry   a   C-­‐terminal   capsid-­‐binding   domain.   Whereas   most   of   TRIM5   proteins   bear   a   C-­‐terminal   PRYSPRY   domain,   some   primate   species   carry   a   Cyclophilin   A   (CypA)   module.  

The  linker  2  (L2)  separates  the  CC  from  the  C  terminal  domain  of  TRIM5  proteins.  

 

 The  PRYSPRY  is  found  on  other  TRIM5  paralogues  and  structural  studies  have   revealed   that   it   forms   a   dimer   interface   via   a   donor   sequence   and   an   acceptor   strand   from   different   protein   targets  268.   This   interface   is   composed   of   six   variable  loops  (VLs)  that  mediate  the  binding  to  different  specific  substrates  269.   Importantly,   the   PRYSPRY   domain   of   TRIM21   recognizes   immunoglobulin   G   (IgG)   with   contacting   residues   found   in   the   VL4,   where   the   E405   and   E406   of   TRIM5α  conferring  species-­‐specific  N-­‐MLV  activity  are  positioned  269,270.  

 

 For   its   part,   the   specificity   of   the   restriction   of   HIV-­‐1   by   rhesus   macaque   TRIM5α   involves   residues   in   the   VL1  271.   Indeed,   the   PRYSPRY   domain   is   the   determinant  of  TRIM5α  retroviral  restriction  specificity  271-­‐273  and  directly  binds   to  the  retroviral  capsid  274,275.  

 

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 The   binding   between   TRIM5α   and   particulate   capsid   protein   was   never   observed.   Notably,   whereas   mature   virions   could   saturate   the   restriction   phenotype,   monomeric   capsid   did   not   have   any   effect   on   retroviral   blockade  

199,237,276,277.  It  became  later  evident  that  the  restriction  factor  recognized  the  CA   in  complexes  forming  an  hexameric  lattice  271,274.  Soon  after  viral  entry  into  the   host  cell  cytoplasm,  TRIM5α  orthologues  bind  to  the  retroviral  capsid  62,200  and   can  subsequently  mediate  a  blockade  at  two  steps  of  the  viral  life  cycle.  

 

 The   use   of   single-­‐cycle   infection   assays   allows   a   safe   and   precise   way   to   determine   what   steps   of   the   viral   life   cycle   are   affected   by   a   cellular   factor   in   non-­‐permissive  (restrictive)  cells.  This  method  uses  combinations  of  three-­‐part   or  two-­‐part  vectors  composed  of  the  viral  genome,  the  packaging  genes  and  an   envelope.  Once  expressed  in  producer  cells,  the  viral  proteins  form  particles  that   are  used  to  infect  target  cells.  The  challenging  of  target  cells  with  these  virions   will   result   in   a   single-­‐round   of   infection,   given   that   no   complete   retroviral   genome  is  provided  and  thus  the  virus  is  replication-­‐incompetent.  Cells  can  also   be  transduced  (figure  10),  referring  to  the  transfer  of  DNA  by  retroviral  vectors.  

When  using  bi-­‐cistronic  vectors  coding  for  a  gene  of  interest  and  for  an  antibiotic   resistance  gene,  cells  can  be  selected  with  the  specific  antibiotic  and  give  rise  to   stable   cell   lines   expressing   constitutively   the   gene   which   function   wants   to   be   studied.  

 

 First,   TRIM5α   impedes   reverse-­‐transcription   to   proceed,   as   revealed   by   the   comparison  of  viral  cDNA  accumulation  between  permissive  and  non-­‐permissive   cell   lines  194,195,202,277.   Indeed,   the   quantification   of   the   reverse-­‐transcripts   in   HeLa  cells  transduced  with  a  bi-­‐cistronic  vector  revealed  that  the  cells  that  stably   expressed   rhesus   TRIM5α   contained   at   least   ten   fold   less   early   and   late   HIV-­‐1   cDNA  products  than  the  cells  that  had  been  transduced  with  the  vector  that  only   carries  the  antibiotic  resistance  gene  62,200.  

 

 The  TRIM5  gene  has  been  subjected  to  strong  positive  selection  on  residues  of   the  PRYSPRY  domain  278,279.  In  at  least  two  independent  events,  the  cyclophilin  A   (CypA)   cDNA   has   inserted   between   exons   7   and   8   or   in   the   3’   Untranslated  

region   (3’UTR)   of   the   TRIM5   gene   via   a   LINE-­‐1-­‐mediated   retrotransposition,   replacing  the  PRY-­‐SPRY  domain  and  forming  a  fusion  protein  (TRIM5Cyp)  62,280-­‐

283   (figure   8).   Whereas   in   the   case   of   the   New   world   monkey  Aotus  trivirgatus   (owl   monkey),   TRIM5Cyp   potently   blocks   HIV-­‐1,   FIV   and   SIV   from   the   African   green  monkey  (SIVagm),  the  version  from  Macaca  mulata  restricts  HIV-­‐2  and  FIV  

62,282,284.    

 Similar  to  TRIM5α,  owl  monkey  TRIM5Cyp  binds  to  the  retroviral  capsid  soon   after  entry  and  blocks  the  reverse-­‐transcription  62,285,286.  The  cylophilin  A  (CypA)   domain   is   responsible   for   the   binding   to   HIV-­‐1   capsid,   as   evidenced   by   the   examination   of   the   effect   of   deletion   mutants   or   the   addition   of   CsA  285,287,288.   Although   it   was   shown   that   the   monomeric   CA   protein   p24   could   bind   to   cyclophilins   A   and   B  289,   the   saturation   of   the   TRIM5Cyp-­‐mediated   blockade   requires   completely   processed   virion   cores  68   indicating   that   the   assembled   hexameric  capsid  is  the  target  of  the  restriction  factor.  

 

 TRIM5  proteins  can  additionally  target  a  second  and  later  step  of  the  retroviral   life  cycle,  as  evidenced  by  the  treatment  of  non-­‐permissive  cells  with  proteasome   inhibitors  such  as  MG132  290-­‐293.  Indeed,  after  addition  of  MG132,  an  increase  in   reverse-­‐transcripts  was  observed,  but  the  restriction  was  still  not  affected.  More   profound  examination  of  the  abundance  of  different  viral  products  showed  that   2-­‐LTR  circles,  a  marker  for  nuclear  import,  were  affected  291,292,  suggesting  that  a   step  before  integration  is  also  affected  by  TRIM5  orthologues.    

 

 The  previous  findings  allow  establishing  a  two-­‐step  model  of  TRIM5-­‐mediated   retroviral   blockade   (figure   7).   First,   inhibition   of   the   reverse-­‐transcription   is   concomitant   with   the   observed   proteasome-­‐dependent   TRIM5-­‐mediated   disassembly  of  the  capsid  290,293.  In  the  second  step,  a  proteasome-­‐independent   mechanism   is   responsible   for   a   block   before,   or   at,   retroviral   cDNA   nuclear   import.  

   

 

1.2.5  TRIM5  is  a  PRR    

 The  expression  of  many  TRIM  proteins  has  been  induced  upon  type  I  IFN  or  TLR   agonist   treatment  262-­‐264,294,295.   Importantly,   TRIM5α   and   TRIM5Cyp   transcripts   are  up-­‐regulated  following  the  addition  of  Type  I  IFN  or  LPS,  in  cells  expressing   the   corresponding   receptors   262,263,296.   Moreover,   we   found   that   TRIM5   is   required  for  the  establishment  of  the  TLR4-­‐mediated  antiviral  state  265.  

 

 In  the  same  study,  we  showed  that  TRIM5  function  as  a  PRR  for  the  retroviral   core.   Notably,   TRIM5   synthesizes   unanchored   K63-­‐polyUb   chains   that   activate   the   TAK1-­‐TAB2-­‐TAB3   complex   leading   to   the   stimulation   of   MAPK-­‐   and   NFκB-­‐

mediated  signaling.  Likewise,  TRIM5Cyp  was  additionally  able  to  stimulate  AP-­‐1   and  NFκB  promoters.  This  activity  of  TRIM5  alone  is  enhanced  when  it  binds  a   restriction-­‐sensitive  capsid.  Furthermore,  the  specific  E2  ligase  Ubc13  and  TAK1   were   found   to   be   necessary   for   TRIM5-­‐mediated   restriction  265.   In   agreement   with  these  findings,  another  study  revealed  the  importance  of  the  E3  ubiquitin   ligase  function  to  the  TRIM5-­‐mediated  retroviral  blockade,  although  in  contrast   with   the   previous   study,   autoubiquitination   was   the   proposed   limiting   process  

247.  Conversely,  a  study  showed  that  the  E3  ligase  function  of  some  TRIM5Cyp  is   not  required  for  retroviral  blockade,  as  evidenced  by  the  conserved  restriction  

247.  Conversely,  a  study  showed  that  the  E3  ligase  function  of  some  TRIM5Cyp  is   not  required  for  retroviral  blockade,  as  evidenced  by  the  conserved  restriction