HIV Attachment & Entry: Insights into pathogenesis and therapeutic

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HIV Attachment & Entry: Insights into pathogenesis and therapeutic Powered By Docstoc
					          HIV
Cellular Pathogenesis II

     Benhur Lee, M.D.
HIV Accessory Genes:
Tat                    Essential in vitro and in vivo
Rev
                       Essential in certain cell types
Vif
                       (Permissive vs Non-permissive cells)
Vpr                    Non-essential in vitro, but leads to
Vpu                    attenuated phenotype in vivo
Nef
Nef
• Major determinant of pathogenicity in
  vivo
  – nef-deleted SIV is severely attenuated in the rhesus macaque
    model
  – infection of macaques with recombinant SIV carrying a
    premature STOP codon (point mutation) results in rapid
    revertants with the nef ORF
  – Patients infected with nef-defective HIV have a dramatically
    decreased rate of disease progression (>15 years)
  – nef-deleted HIV do not deplete thymocytes as much, or
    replicate to as high titers, as wild-type HIV in the SCID-
    hu mice model
        Pleiotropic Functions of Nef
  N-myristoylation required for
  Nef activity--implies that membrane
  localization of nef is critical
  for its activity

Consensus
N-myristoylation
Signal:            MGxxx(S/T)(K/R)(K/R)



HIV sequence    MGxxx(S)(K)(K/R)
Conservation in
Nef protein:    100%    99%
                     100% ~50%
       Pleiotropic Functions of Nef
• Down-regulates cell surface levels
  of CD4

• Down-regulates surface levels of
  major histocompatibility class I
  molecules

• Mediates cellular signaling and
  activation

• Enhances viral infectivity
    I. Down-modulation of surface CD4
•   Down-modulation of surface CD4 via internalization
    followed by degradation via endosomal/lysosomal
    pathway

•   Advantages:
     –   Prevents disadvantageous super-infection of
         host cell
     –   Enhance viral progeny release (by preventing
         Env-mediated sequestration of CD4 in secretory
         pathway)

•   Evidence:
     –   Nef expression increases number of CD4
         containing clathrin-coated pits
     –   Nef-induced CD4 down-modulation blocked by                              ..
         inhibitors of clathrin-coated pit-mediated                    .............
                                                                        . . . ..
                                                                           .
         endocytosis (e.g. ikaguramycin)                             ................... ...
                                                                        ....... .
                                                                           .
                                                                      .. . . . . .. .
     –   Inhibition of lysosomal acidification (e.g. via                                . .
                                                                       .. .. .. ............. .
                                                                                     ................
         chloroqine treatment) blocks Nef-induced CD4          CD4                    .. . ... .
                                                                                    ..... ...
                                                                                    . . ... .. ..
         degradation                                                                     ... ........
     –   Expression of nef alone in T-cell lines can lead to                                 ..........
                                                                                               . ...
         CD4 downregulation (as determined by FACS)                                                 .
                                                                          Nef-GFP
    I. Down-modulation of surface CD4
•   Mechanism(s)?
    –   Direct interaction with CD4 has not been biochemically
        demonstrable, but NMR analysis suggest a direct
        interaction with Kd ~0.87 mM; yeast two-hybrid assays
        also suggest an interaction

    –   Acts as a connector to the host-cell endocytic
        machinery
          • Co-localizes with AP-2 on inner plasma membrane
          • Conserved dileucine based sorting motif
             (E/DxxxL ) in Nef is important for both CD4-down-
             modulation and AP-2 co-localization
          • Interacts with NBP-1 (identified through a yeast
             two-hybrid screen). NBP-1 is part of the vacuolar
             membrane ATPase complex in clathrin-coated pits        NBP-1
             (H subunit of vacuolar ATPase--VH1)
          • C-terminal diacidic motif (DD) in Nef is important
             for NBP-1 interaction, and, at least in SIV Nef, the
             dileucine motif is also important for NBP-1
             interactions
          • ?? May bind to b-Cop, a coatamer protein which
             targets proteins to lysosomes
  II.Down-modulation of MHC Class I

• Advantages:
   – Immune evasion; MHC Class I presents
     antigens to cytotoxic T- lymphocytes; alerts
     innate and adaptive immune system to
     virally infected cells
• Evidence:
   –   Nef expression reduces susceptibility of HIV-
       infected cells to CTL mediated lysis in vitro
   –   selectively down-regulates only HLA-A and HLA-
       B, which presents antigens to CTLs;
   –   does NOT down-regulate HLA-C and HLA-E,
       which inhibit NK-cell mediated cell lysis
   –   Thus, efficiency of CTL-mediated lysis (adaptive
       immunity) is reduced without increasing
                                                                 CTL
       increasing susceptibility to NK cell lysis

 HIV                                                                    HIV antigen
                                                                   MHC Class I
                                                          51Cr
  100%                              HIV Dnef
                                    HIV wt



% Lysis

                                               E (Effector Cell)

                                                   CTL
    0%
          1:2   1:5   1:10   1:20                           HIV antigen
                                                       MHC Class I
                 E:T ratio



                                               T (Target Cell)
      III. Mediates Cellular Activation and
                    Signaling

•   Nef expression upregulates a transcriptional program that
    activates the HIV LTR (microarray analysis)
      III. Mediates Cellular Activation and
                    Signaling

•   Nef expression upregulates a transcriptional program that
    activates the HIV LTR (microarray analysis)

•   Nef can induce secretion of paracrine factors that enhance viral
    replication; macrophage supernatants from cells transduced with
    nef-expressing adenoviral vector can facilitate HIV replication in
    resting lymphoid cultures

                                                 Adv-nef supnt
  p24                                            Adv-GFP supnt
(ng/ml)



                3      6      9 (days)
      III. Mediates Cellular Activation and
                    Signaling

•   Nef expression upregulates a transcriptional program that
    activates the HIV LTR (microarray analysis)

•   Nef can induce secretion of paracrine factors that enhance viral
    replication; macrophage supernatants from cells transduced with
    nef-expressing adenoviral vector can facilitate HIV replication in
    resting lymphoid cultures

•   Nef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2
    complex may regulate many of Nef’s effect on gene transcription
   IV. Infectivity Enhancement
• Magnitude of infectivity enhancement is
  allele dependent
• Nef mediated enhancement can be
  provided in trans
  – reporter gene (e.g. GFP or luciferase) expression under control
    of the LTR promoter can be enhanced when nef expression
    vector is co-transfected
• Mechanisms:
  – Increased RT activity; increased proviral DNA
    synthesis
  – Increased cytoplasmic delivery of viral
    particles
      Vif: Viral infectivity factor, required for robust
           replication only in certain cells




                   C8166, 293T,               Hut78, H9,
                   HeLa                       1º PBLs
                   Permissive               Non-permissive
HIV-1 (           +++ replication           +++ replication

HIV-1 (Dvif)       +++ replication           no replication

Two hypotheses:
(a) Permissive cells express an activity (factor) that can
    compensate for vif.
(b) Non-permissive cells have an inhibitory activity on
    viral replication, which is overcomed by vif.

                           See Simon et. al., Nature Med. 4: 1397
Non-permissive         Permissive           Heterokaryon


       Dvif            wt    Dvif            wt       Dvif
  wt




                                     Which phenotype will dominate?

                                     Non-permissive: inhibitory cellular
                                     factor overcomed by vif

 ++       -            ++       ++   Permissive: compensatory factor
                                     similar to vif
              Infectivity
                                   Denv vs Denv/Dvif
                                    Non-Permissive     Permissive




                                                           (ensures that
                                                           all viruses are
                                                           produced from
                                                           Heterokaryons)




  Two hypotheses:
(a) Permissive cells express an
     activity (factor) that can
     compensate for vif.
(b) Non-permissive cells have an          Permissive
    inhibitory activity on
    viral replication,
    which is overcomed by vif.
                                            Heterokaryon

Non-permissive         Permissive
                                              wt          Dvif

  wt   Dvif            wt    Dvif




                                              ++            -
 ++       -            ++       ++   Non-permissive: inhibitory cellular
                                     factor overcomed by vif
              Infectivity
                                                           Expression of CEM15 in
                                                           CEM-SS (permissive cells)
                                                           renders it non-permissive




After subtractive-PCR for differentially expressed genes
              CEM15=APOBEC3G
   (Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide-like 3G)
• APOBEC3G, cytosine
  deaminase
   –   APOBEC1, cytidine deaminase
       involved in mRNA editing
   –   AID (Activation-induced cytidine
       deaminase), involved in somatic
       hypermutation, required for
       immunoglobulin gene diversification
• Incorporated into virions
   – Deaminates cytosines to uracils
     during first strand cDNA
     synthesis in target cells
   – Moderate sequence specificity Degradation: uracil in DNA recognized
     (Py-C-C)                        by host uracil DNA glycosidase, which
                                     removes uracil from DNA-->cleavage
                                     and degradation
                                     Hypermutation: massive C-->U conversion
                                     on minus strand leads to massive
                                     G-->A hypermutation of the plus strand;
                                     resultant provirus is so heavily mutated,
                                     no functional viral proteins are encoded
How does vif overcome APOBEC3G’s
        antiviral function?
• Reduces the level of
  APOBEC3G in producer
  cell
  – Expression of vif reduces
    APOBEC3G levels
  – Vif promotes degradation of
    APOBEC3G via Ubiquitin-
    proteasome pathway
  – Vif impairs efficient
    translation of APOBEC3G
    mRNA

				
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