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Chapter  1  

 

INTRODUCTION    

1.1.1  Retroviruses  

 Viral   replication   requires   the   transcription   and   translation   machinery,   which   they  themselves  lack,  from  the  organisms  that  they  infect.  

The  common  feature  among  all  retroviruses,  and  also  what  makes  them  unique   among  other  viruses,  is  that  they  reverse  transcribe  their  RNA  genome  into  DNA   that   can   be   inserted   into   the   host   genome   1   (figure   1).   For   this   process,   retroviruses   encode   an   RNA-­‐dependent   DNA   polymerase,   an   RNAseH,   and   a   host-­‐encoded   transfer   RNA   (tRNA)   that   serves   as   the   primer   for   reverse   transcription.  

 

 Discovered   and   first   studied   as   disease-­‐causing   agents,   many   decades   of   research  has  uncovered  the  molecular  mechanisms  governing  spread,  replication   and  disease  progression  caused  by  these  viruses.  

Retroviruses  can  produce  fast  and  slow-­‐progression  diseases  including  various   types  of  tumors  and  immunodeficiency  2,3.  

 

   A   principal   characteristic   of   these   viruses   is   that   they   integrate   into   the   host   genome  4.   This   is   the   reason   why,   they   later   became   to   be   used   as   vectors   for   gene  delivery  into  cells  5-­‐7.  

   

If  the  integration  event  happens  in  the  germline,  the  retroviral  sequences  can  be   spread   from   one   generation   to   the   next,   a   phenomenon   called   vertical   transmission8.  This  feature  of  retroviruses  is  exploited  to  trace  the  evolution  of   genes   of   the   host   species,   which   inherits   retroviral   sequences   in   a   Mendelian   fashion,  and  used  in  the  study  of  speciation  9,10.  

 

 A  subset  of  retroviruses  lead  to  cell  transformation  and  cancer,  therefore  they   are   designated   as   oncoviruses   11.   The   investigation   of   the   corresponding   insertion   sites   within   the   genome   thus   lead   to   the   identification   of   genes   involved   in   cell   growth   and   tumor   promotion   12.   Certain   oncoviruses   have   inherent  transforming  potential  owing  to  the  prior  acquisition  of  host  sequences  

13This   characteristic   is   useful   for   the   study   of   genetic   regulation   of   the   cell   growth  14.  

 

   

Figure  1:  The  retroviral  life  cycle.  The  main  steps  of  the  retroviral  replication  cycle  are  depicted.  Blue:  

capsid;  yellow:  nucleocapsid;  black  bars  within  the  nucleocapsid:  RNA  genome;  orange  bars:  DNA  genome.  

Courtesy  of  Prof.  Jeremy  Luban  (adapted).  

 

1.1.2  The  discovery  of  retroviruses  and  the  reverse-­‐transcriptase  enzyme      

 The  first  retroviruses  were  discovered  at  the  beginning  of  the  twentieth  century   as  oncogenic  agents  affecting  birds.  Ellermann  and  Bang  found  that  leukosis  in   poultry  was  caused  by  a  factor  present  in  ultra-­‐filtered  cell  extracts  15,  that  was   later  called  Avian  Leukemia  virus.  A  few  years  later  Rous  showed  that  an  agent  

Expression Assembly

Nuclear transport Budding Maturation Binding

Membrane Fusion

Reverse Transcription

Integration

present  in  cell-­‐free  extracts  was  the  intrinsic  cause  of  the  sarcoma  formation  in   the   fowl  5,16.   This   retrovirus   was   named   after   the   man   who   discovered   it,   the   Rous  Sarcoma  Virus  (RSV).  

 

 Almost  thirty  years  later,  a  murine  virus  was  found  to  be  the  agent  provoking   leukemia  in  mice  and  thus  was  called  Murine  Leukemia  Virus  (MLV)  17.  

MLV  belongs  to  the  gammaretrovirus  genus  of  the  retroviridae  family  17.    

 The  concept  of  an  RNA  virus  converting  its  genetic  material  into  a  DNA  form  and   integrating  into  the  host  genome  was  not  yet  formulated.  The  only  information   scientists   had   at   that   moment   was   that   the   viral   agent   did   not   have   a   DNA   genome  but  instead  was  constituted  of  RNA  5.  

 

 In   the   beginning   of   the   1960s,   the   molecular   biologist   Howard   Temin   worked   with   the   RSV   and   found   that   inhibiting   the   DNA   synthesis   blocked   the   viral   replication  18.   This   led   him   to   propose   the   provirus   hypothesis.   That   is,   the   retroviruses  have  a  DNA  intermediate  in  the  cells  that  they  infect.  

 

 Later,   in   the   year   1970   his   team   and   another   virologist   involved   in   the   MLV   research  published  separately  data  showing  the  presence  in  RNA  tumour  virus   particles   -­‐   called   virions   -­‐   of   a   RNA-­‐dependent   DNA   polymerase   activity,   by   correlating  the  induced  RNA  degradation  with  the  decrease  of  the  DNA  synthesis.    

 This  enzyme  was  later  called  the  reverse-­‐transcriptase  13,19,20.      

 In  humans,  certain  types  of  acute  leukemias  were  studied  and  the  viral  cause  of   this  disease  was  soon  investigated.  A  type  C  morphology  (that  will  be  defined  in   the   following   section)   retrovirus,   which   close   relative   had   been   discovered   to   induce   leukemia   in   the   Gibbon   ape   was   pointed   out   by   Robert   Gallo   to   be   the   cause   of   the   human   disease   and   it   was   called   Human   T   cell   Leukemia   Virus   (HTLV)  21.  

 

 The   previous   findings   provided   the   biochemical   and   molecular   tools   that   ultimately   allowed   the   subsequent   identification   of   the   Human  

Immunodeficiency   Virus   type   1   (HIV)   as   the   agent   causing   the   Acquired   Immunodeficiency  Syndrome  (AIDS)  22.  

In  the  next  section,  I  will  introduce  the  general  retroviral  structure.  

 

1.1.3  The  general  structure  of  retroviruses    

 It   all   starts   with   the   viral   RNA.   The   positive   single-­‐stranded   RNA   genome   is   composed  of  different  regulatory  sequences  and  open  reading  frames  (ORFs)  12   (figure  2),  and  has  a  5’  cap  and  a  poly  A  tail.  

The   regulatory   elements   are   located   at   the   extremities   of   the   viral   RNA   and   consist   of   repeated   (R)   sequences,   a   unique   5’   sequence   (U5)   containing   a   cis-­‐

acting  attachement  (att)  site,  a  unique  3’  sequence  (U3),  the  primer  binding  site   (PBS),   the   psi   (packaging   signal)   element   (ψ)   and   a   polypurine   tract   (PPT)  12   (figure  2).  

 

 The   R   regions   are   redundant   in   sequence   and   are   found   after   the   m7G5’ppp5’Gmp   cap,   which   mimics   the   eukaryotic   mRNA   5’cap.   The   U5   sequence  is  immediately  downstream  of  the  5’  R  sequence  and  contains  the  att   sequence  that  is  involved  in  proviral  integration.  These  regions  are  followed  by   the  PBS  where  the  specific  tRNA  primer  hybridizes  and  starts  the  transcription   of  the  minus-­‐strand  DNA  (-­‐sDNA).  The  next  sequence  in  the  RNA  genome  is  the  ψ   region  recapitulating  most  of  the  sequences  required  for  viral  genome  packaging   into   the   viral   particles.   A   major   splice   donor   site,   that   gives   rise   to   different   subgenomic   mRNAs,   often   closely   follows   this   element.   Subgenomic   RNAs   are   different   mRNA   species   created   when   reverse   transcription   jumps   on   the   template  in  the  3’  to  5’  orientation.  The  resulting  mRNAs  have  variable  5’  regions   overlapping   with   the   template   strands   at   different   levels   but   the   same   3’  

sequence.  The  generation  of  various  mRNAs  allows  condensing  a  high  amount  of   information  12.  

 

 The   PPT,   positioned   at   the   3’   end   of   the   viral   genome,   consists   of   a   row   of   purines   Adenine   and   Guanosine,   required   for   the   initiation   of   the   +sDNA   transcription.  

Finally,   the   U3   region   preceding   the   polyA   tail   contains   another   att   site   and   in   addition  a  set  of  cis-­‐regulatory  sequences  essential  for  viral  gene  expression.  

Given  that  the  synthesis  of  the  viral  DNA  involves  a  duplication  of  the  extremities   of  the  RNA  templates  with  a  subsequent  transfer  of  the  U5  and  U3  regions,  the   two  ends  in  the  resulting  dsDNA  are  identical  and  these  are  called  Long  Terminal   Repeats  (LTRs)  12.  

   

The  provirus  is  integrated  and  found  in  the  host  genome  with  the  flanking  LTRs  

5.   When   the   provirus   is   transcribed,   the   5’   U3   region   is   not   taken   into   account   and  the  synthesis  proceeds  until  the  R  to  U5  boundary.  In  this  way,  the  resulting   viral   RNA   has   the   same   genomic   organization   as   the   template   from   viral   particles.  

 

 The  viral  proteins  are  encoded  by  three  ORFs,  namely  the  group  antigen  (gag),   the   polymerase   (pol)   and   the   envelope   (env).   These   genes   code   for   precursos   that  once  cleaved  will  give  rise  to  more  than  one  protein.    

The  gag   ORF   codes   for   the   matrix   (MA),   the   capsid   (CA)   and   the   nucleocapsid   (NC)  12.  

The  pol  gene  products  are  the  protease  (PR),  the  reverse-­‐transcriptase  (RT),  the   integrase  (IN)  and,  in  some  cases,  a  dUTPase.  

 

 Finally,  the  precursor  synthesized  from  the  env  gene  is  cleaved  into  the  surface   envelope  protein  (SU)  and  the  transmembrane  envelope  protein  (TM)  12.  

 

 Once  processed  from  their  precursors  the  viral  proteins  form  the  mature  virion,   which  is  able  to  infect  susceptible  cells  that  express  the  appropriate  receptors.  

The  viral  core  of  a  mature  viral  particle  consists  in  the  diploid  RNA  genome  that   interacts   with   the   NC,   creating   a   condensation,   surrounded   by   the   CA   protein   complex.  The  matrix  protein  that  covers  this  core  is  surrounded  on  top  by  a  host-­‐

derived  lipid  bilayer  and  the  included  SU  and  TM  proteins  12    

 The  viral  core  contains  as  well  the  pol-­‐derived  proteins  that  will  be  used  for  a   novel  round  of  replication,  namely  the  PR,  the  RT  and  the  IN  12.  

   

 

Figure   2:   Schematic   view   of   the   proviral   genome   structure   of   retroviruses.   The   retrovirus   proviral   DNA  is  composed  of  untranslated  regions  that  flank  the  ORFs  for  gag,  pro,  pol,  env  and  in  some  cases  that  of   accessory   genes.   The   flanking   LTRs   contain   U3   and   U5   regions,   as   well   as   a   repeat   sequence   (R).   The   5’  

region  of  the  retroviral  genome  is  followed  by  a  PBS  and  a  psi  encapsidation  signal.  Adjacent  to  the  last  ORF,   the  viral  RNA  contain  a  PPT.  ORF:  open-­‐reading  frame;  LTR:  log  terminal  repeats;  U3  and  U5:  unique  regions   3  and  5,  respectively;  att:  attachemetn  site;  PBS:  primer  binding  site;  PPT:  poly-­‐purine  tract.  Adapted  from   Fouty  and  Solodushko,  2011  23  .  

 

1.1.4  The  reverse-­‐transcription  process.  

 Once  the  retroviral  genome  enters  the  cell,  the  diploid  single-­‐stranded  genome   that   is   still   bound   to   the   nucleocapsid   (NC)   protein,   constituting   the   viral   core,   starts  the  process  of  reverse  transcription  24,25.  

 

 For   reverse   transcription   to   take   place,   important   elements   contained   in   the   viral  particles  are  required.  The  central  component  is  the  reverse  transcriptase   enzyme,   which   catalyzes   four   different   reactions:   RNA-­‐dependent   and   DNA-­‐

dependent  DNA  polymerization,  DNA  strand  separation  via  its  helicase  function   and   the   hydrolysis   of   the   RNA   fragments   on   RNA-­‐DNA   heteroduplexes  26.   The   viral   core   carries   additionally   a   specific   collection   of   transfer   RNA   (tRNA)  

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molecules,  different  cellular  messenger  RNAs  (mRNAs)  from  previously  infected   cells  and  some  ribosomal  RNA  (5S  and  7S)  26.  

 

 Reverse  transcription  starts  when  the  3’  region  of  a  specific  tRNA  is  used  as  a   primer  that  anneals  with  the  PBS  within  the  5’  region  of  the  viral  RNA  genome   (figure   3).   DNA   synthesis   continues   until   the   5’   extremity   of   the   RNA   strain   is   reached,  resulting  in  a  short  DNA  strand  called  the  minus  strand  strong  stop  DNA   (–ssDNA)  27.  

 

 The  next  step  takes  the  advantage  that  the  minus-­‐strand  DNA  contains  a  repeat   (R)   sequence   that   is   present   at   both   viral   genome   termini   and   that   was   introduced  in  the  newly  synthesized  DNA  molecule  by  the  reverse  transcription   of  the  5’  region  of  the  viral  RNA.  This  confers  a  complementarity  of  the  –sDNA   and   the   3’   end   of   the   RNA   genome   that   allows   the   transfer   of   the   small   oligonucleotide   to   that   region,   after   that   the   RNAse   H   function   of   the   RT   has   degraded  the  RNA  to  which  the  newly  synthesized  DNA  is  annealed.  This  marks   the  beginning  of  the  elongation  of  the  –sDNA  chain,  with  an  accompanying  RNA   degradation  accomplished  by  RNAse  H  27.  

 

 During   the   RNA   dependent-­‐DNA   synthesis,   the   ppt   permit   the   RNA   to   escape   degradation  and  this  RNA  fragment  is  then  used  as  a  primer  for  the  plus-­‐strand   DNA  (+sDNA)  polymerization  that  finally  reaches  the  U5  region  of  the  –sDNA.  In   the   mean   time,   the   –sDNA   continues   to   be   polymerized,   with   a   subsequent   gradual  RNA  degradation.  

 

 In   the   following   step,   the   +sDNA   synthesis   proceeds   until   the   level   of   the   PBS   complementarity  is  formed  and  the  RNA  and  tRNA  primers  are  degraded.  When   the  tRNA  is  removed  from  the  +sDNA  a  complementarity  region  is  exposed  and   the   second   strand   transfer   happens   where   the   plus   and   minus   strands   anneal.  

The  resulting  molecule  is  a  circular  DNA  intermediate  27.    

 This  point  of  the  viral  replication  cycle  can  lead  to  a  non-­‐productive  dead-­‐end   DNA  molecule  which  contains  a  single  LTR  or  to  a  productive  DNA  form  flanked  

by   two   LTRs,   resulting   from   the   strand   displacement   of   the   plus   and   minus  

1.1.5  The  classification  of  retroviruses     50end of the viral RNA, exposing the newly syn-thesized minus-strand DNA (see Fig. 1).

The ends of the viral RNA are direct repeats, called R. These repeats act as a bridge that allows the newly synthesized minus-strand DNA to be transferred to the 30end of the viral RNA. Retro-viruses package two copies of the viral RNA

genome; the first (or minus-strand) transfer can involve the R sequence at the 30ends of either of the two RNAs (Panganiban and Fiore 1988; Hu and Temin 1990b; van Wamel and Berkhout 1998; Yu et al. 1998). After this trans-fer, minus-strand synthesis can continue along the length of the genome. As DNA synthesis proceeds, so does RNase H degradation. How-ever, there is a purine-rich sequence in the RNA genome, called the polypurine tract, or ppt, that is resistant to RNase H cleavage and serves as the primer for the initiation of the

R U5 pbs gag pol env ppt U3 R

Figure 1.Conversion of the single-stranded RNA genome of a retrovirus into double-stranded DNA. (A) The RNA genome of a retrovirus (light blue) with a tRNA primer base paired near the 50end. (B) RT has initiated reverse transcription, generating minus-strand DNA (dark blue), and the RNase H activity of RT has degraded the RNA template (dashed line). (C) Minus-strand transfer has occurred between the R sequences at both ends of the genome (see text), allowing minus-strand DNA synthesis to continue (D), accompanied by RNA degradation. A purine-rich sequence (ppt), adjacent to U3, is resistant to RNase H cleavage and serves as the primer for the synthesis of plus-strand DNA (E). Plus-strand synthesis continues until the first 18 nucleotides of the tRNA are copied, allowing RNase H cleavage to remove the tRNA primer. Most retroviruses remove the entire tRNA; the RNase H of HIV-1 RT leaves the rA from the 30end of the tRNA attached to minus-strand DNA. Removal of the tRNA primer sets the stage for the second ( plus-strand) transfer (F); extension of the plus and minus strands leads to the synthesis of the complete double-stranded linear viral DNA (G).

HIV-1 Reverse Transcription

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retroviruses   consist   of   the   Alpharetroviruses,   Betaretroviruses   and   Gammaretroviruses  12.  

 

 Alpharetroviruses   infects   a   large   range   of   birds.   They   assemble   at   the   cell   membrane  and  possess  a  central  spherical  core  (C-­‐type  morphology).  The  tRNA   they   use   for   the   priming   of   reverse   transcription   is   the   one   for   tryptophan   (tRNATrp).  A  typically  well-­‐studied  member  of  this  genius  is  the  Avian  Leukosis   Virus  (ALV)  and  the  previously  mentioned  RSV  12.  

 

 Members  of  the  Betaretroviruses  infect  different  mammalian  species  including   mice  and  primates.  Morphologically,  they  can  have  either  an  asymmetric  round   core,  either  a  cylindrical  one.  They  contain  a  dUTPase  gene  in  frame  with  the  pro   gene   and   they   use   the   tRNALys.   The   oncovirus   Mouse   Mammary   Tumor   Virus   (MMTV)  is  a  member  of  this  family  12.  

 

 Gammaretroviruses   possess   C-­‐type   virion   morphology.   They   have   two   ORFs.  

The   first   one   encodes   the  gag,  pro   and  pol   gene   products;   the   second   one   encodes  the  envelope  proteins.  The  tRNAs  used  by  these  retroviruses  are  mainly   the  ones  for  proline  or  glutamine.  Highly  documented  oncogenic  members  of  this   genius  include  the  Murine  Leukemia  Virus  (MLV),  Feline  Leukemia  Virus  (FLV)   and  Gibbon  Ape  Leukemia  Virus  (GALV)  12.  

 

 The   group   of   complex   retroviruses   is   composed   of   Deltaretroviruses,   Epsilonretroviruses,   Lentiviruses   and   Spumaviruses.   Deltaretroviruses   and   Epsilonretroviruses  have  a  similar  C-­‐type  virion  morphology.  The  first  genius  is   composed   of   members   encoding   two   accessory   proteins   named   rex   and   tax,   which   are   involved   in   the   synthesis   and   processing   of   viral   RNA.   It   uses   the   tRNAPro.  An  example  of  this  group  is  the  oncovirus  Human  T-­‐Lymphotropic  Virus   1  (HTLV-­‐1)  and  the  closely  related  HTLV-­‐2.  The  second  genius  is  uses  the  tRNA   for  histidine  or  arginine  and  codes  additionally  for  three  proteins  called  ORFA,  B   and   C   respectively.   The   function   of   these   accessory   proteins   are   not   well   understood   but   in   the   case   of   the   better-­‐studied   member   Walley   Dermal   Sarcoma   Virus   (WDSV),   ORFA   has   been   shown   to   be   an   orthologue   of  

mammalian   cyclin   c,   ORFB   activates   the   PKC   and   AKT   signaling   and   ORFC   has   oncolytic  properties  29.  

 

 The   AIDS-­‐causing   HIV-­‐1   belongs   to   the   genius   of   lentiviruses   and   is   characterized   by   a   conical   shape   of   the   core   of   the   mature   virion.   Members   of   this  group  carry  this  name  because  of  the  long  asymptomatic  phase  preceeding   the   first   symptoms   12.   HIV-­‐1   expresses   six   accessory   proteins   that   will   be   discussed  below.  These  gene  products  control  transcription,  gene  expression  and   assembly  and  counteract  restriction  factors  encoded  by  the  host  12.  The  primer   used  by  lentiviruses  is  the  tRNALys3.  

 

 In   latin   Spuma   means   foam.   The   members   of   the   Spumaviruses   produce   vacuolization   of   cells,   hence   resulting   in   a   foamy-­‐like   histological   aspect.   The   human   foamy   virus   is   a   well-­‐studied   member   of   this   group.     The   pol   gene   products  arise  from  a  splice  transcript.  Unlike  other  retroviruses,  this  genius  of   viruses   is   characterized   by   virions   that   carry   high   amounts   of   reverse-­‐

transcribed   DNA.   Accessory   proteins   shared   by   the   members   of   this   group   include  a  transcriptional  transactivator.  The  primer  used  is  generally  tRNALys  12.    

1.1.6  The  Acquired  Immunodeficiency  Syndrome  (AIDS)    

   The  AIDS  is  a  severe  disease  affecting  more  than  35  millions  of  people  around   the  world,  as  published  by  the  UNAIDS  report  on  the  global  AIDS  epidemics  2013  

30.    

 In  the  early  1980s,  young  men  with  typical  immunodeficiency  symptoms  were   hospitalized  in  Los  Angeles,  New  York  and  California  31,32.  

 

 As   mentioned   previously,   biochemical   and   genetic   tools   for   studying   retroviruses   existed   in   that   decade   and   they   were   used   by   Researchers   at   the   Institut  Pasteur  and  in  the  United  States  to  characterize  the  virus  extracted  from   CD4+  T  cells  coming  from  AIDS  patients.  Barré-­‐Sinoussi  and  colleagues  isolated   and   described   a   virus   that   was   able   experimentally   to   infect   T   lymphocytes  

extracted  from  cord  blood  22  and  called  it  Lymphoadenopathy  Associated  Virus   (LAV).  

 

 The  team  of  Robert  Gallo,  had  suspected  that  the  causing  agent  of  AIDS  was  of   retroviral  origin  and  possessed  T-­‐cell  tropism  but  at  that  time  attributed  it  to  the   human   tumor   retrovirus   HTLV-­‐I  33.   The   virus   was   later   called   HTLV-­‐III   by   the   same  team.  

In  1986,  the  virus  was  finally  named  the  Human  Immunodeficiency  Virus  (HIV),   in  reference  to  the  disease  it  produced  34.  

 

 Transmission   of   HIV-­‐1   from   one   person   to   another   happens   during   sexual   intercourse,   injecting   with   contaminated   needles,   or   by   blood   transfusion  35.   Mother  to  child  transmission  during  delivery  or  after  breast-­‐feeding  is  another   important  route  of  spreading  35.  

The   first   events   of   HIV-­‐1   infection   seem   to   implicate   a   local   spreading   within   cells  residing  in  the  mucosa  and  in  the  epithelium,  such  as  dendritic  cells  (DCs),   CD4+  T  cells  and  macrophages  36-­‐38.  Primary  infected  cells  subsequently  migrate   to  the  lymphoid  organs  and  seed  the  virus  by  direct  cell-­‐to-­‐cell  contact  or  by  the   release  of  newly  produced  cell-­‐free  viruses,  which  enter  new  cells  39.  

 

 When   HIV-­‐1   gp120/gp41   glycoproteins   interact   with   the   lectin   receptor   DC-­‐

SIGN   at   the   surface   of   DCs,   the   virus   can   be   either   endocytosed   and   degraded   within  lysosomes  or  by  targeting  to  the  proteasome  40,41.  Another  route  for  entry   into   DCs   is   mediated   by   a   host-­‐derived   glycosphingolipid   present   in   the   virion   envelope   that   binds   to   an   unknown   receptor,   with   SIGLEC-­‐1   being   a   potential   candidate  42.   This   interaction   allows   the   virus   to   escape   degradation   and   join   immunological   synapses,   from   where   new   target   CD4+   T   cells   can   be   reached  

12,43,44.    

 During  the  acute  phase  of  infection,  a  large  fraction  of  CD4+  T  cells  are  infected   and  high  amounts  of  virions  are  synthesized  and  released  from  cells  39.  As  CD8+  

T  cells  fight  against  the  pathogen  and  high  doses  of  type  I  interferon  (IFN)  and   cytokines   are   released,   infected   individuals   commonly   experience   flu-­‐like  

symptoms  45-­‐47.  The  immune  response  mediated  by  cytotoxic  T  cells  and  B  cells   producing  antibodies  permits  to  moderately  recover  the  level  CD4+  T  cells  for  a   few  weeks  47.  At  that  point,  HIV-­‐1  already  integrated  into  the  host  chromosomes   and  latent  reservoirs  starts  to  be  established,  and  infected  individuals  can  have  a   total  absence  of  HIV-­‐1-­‐related  symptoms  for  nearly  ten  years  47.  Unfortunately,   in   the   meantime,   the   virus   continues   to   replicate   and   spread   via   the   various   lymphoid  organs.  

 

 At  the  terminal  stage,  the  disease  causes  a  high  destruction  of  the  CD4+  T  cells,   which   decrease   below   200   cells   per   mm3   of   blood,   leading   to   immune   suppression   and   the   subsequent   unavoidable   infection   by   opportunistic  

 At  the  terminal  stage,  the  disease  causes  a  high  destruction  of  the  CD4+  T  cells,   which   decrease   below   200   cells   per   mm3   of   blood,   leading   to   immune   suppression   and   the   subsequent   unavoidable   infection   by   opportunistic