HIV Cellular Pathogenesis II

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HIVCellular Pathogenesis II

• Benhur Lee, M.D.

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HIV Accessory Genes Tat Rev Vif Vpr Vpu Nef
Essential in vitro and in vivo
Essential in certain cell types (Permissive vs
Non-permissive cells)
Non-essential in vitro, but leads to attenuated
phenotype in vivo
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Tat Transactivator of HIVs LTR Promoter
2º structure of HIV TAR sequence

• Experimental Observations
• Binding of Tat to TAR in vitro does NOT require
  loop sequences known to be necessary in vivo for
  function
• Pre-incubation of nuclear extracts with
  recombinant Tat depletes a factor necessary for
  Tat-mediated transcription in vitro
• Tat functions poorly in rodent cells unless
  complemented by factor(s) present on Chromosome
  12 (radiation hybrids)
• Tat associates with a kinase complex that
  hyperphosphorylates CTD of RNAP II (identified
  thru an in vitro drug screen for Tat
  inhibitors)--this kinase is Cdk9, but Cdk9 does
  NOT bind Tat!?
• Mystery human-specific co-factor for Tat activity
  must exist

loop
bulge
4

• Predicted and confirmed properties of Tat
  co-factor Cyclin T
• Binds directly to Tat in a complex with Cdk9
• Increases the affinity of Tat for TAR
• Increases the specificity of Tat for loop and
  bulge residues
• Tat-CycT-Cdk9 complex hyperphosphorylates CTD of
  RNAP II
• and increases HIV transcriptional processivity
• CycT maps to chromosome 12, and potentiates
• Tat trans-activation in murine cells 50- to
  100- fold
• Murine homolog of human CycT does NOT bind to Tat

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Tat Transactivator of HIVs LTR Promoter

• Experimental Observations Explained
• Binding of Tat to TAR in vitro does NOT require
  loop sequences known to be necessary in vivo for
  function
• Pre-incubation of nuclear extracts with
  recombinant Tat depletes a factor necessary for
  Tat-mediated transcription in vitro
• Tat functions poorly in rodent cells unless
  complemented by factor(s) present on Chromosome
  12 (radiation hybrids)
• Tat associates with a kinase complex that
  hyperphosphorylates CTD of RNAP II (identified
  thru an in vitro drug screen for Tat
  inhibitors)--this kinase is Cdk9, but Cdk9 does
  NOT bind Tat!?
• Mystery human-specific co-factor for Tat activity
  must exist Cyclin T

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Rev

• Essential for nuclear export of unspliced or
  single spliced viral transcripts

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Arg Rich Domain (ARD) --binds to Importin-b for
nuclear import
After nuclear import, Ran-GDP is converted to
Ran-GTP, and importin- b dissociates from Rev
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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 (gt15 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

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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)
MGxxx(S)(K)(K/R)
HIV sequence Conservation in Nef protein
99
100
50
100
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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

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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
  degradation
• Expression of nef alone in T-cell lines can lead
  to CD4 downregulation (as determined by FACS)

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CD4
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Nef-GFP
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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
  (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

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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
  increasing susceptibility to NK cell lysis

HIV
51Cr
14
100
HIV Dnef
HIV wt
Lysis
E (Effector Cell)
0
12
15
120
110
ET ratio
T (Target Cell)
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III. Mediates Cellular Activation and Signaling

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

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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 (ng/ml)
Adv-GFP supnt
3
6
9
(days)
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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 Nefs
  effect on gene transcription

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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

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Vpu CD4 down-modulation

• 16 kDa, membrane spanning
• Binds CD4 tail in the ER
• Targets CD4 for proteolysis via
• ubiquitin-proteasome pathway

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Vpu mediated CD4 degradation via
ubiquitin-proteasome pathway

• Evidence
• Vpu activity disrupted by
• inhibitors of proteasome-
• mediated proteolysis
• Vpu activity affected by
• dominant negative mutants
• of ubiquitin pathway
• Removal of lysine residues
• (ubiquination targets) in CD4 tail
• prevents Vpu-mediated degradation
• Vpu binds to b-TrCP, which in turns
• binds to the proteasome targeting
• factor Skp1p
• Overexpression of b-TrCP mutant
• that cannot bind Skp1p inhibits
• Vpu-mediated CD4 degradation

Contrast with Nef
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Vpu required for proper maturation and targeting
of progeny virions, and for their
proper release from the cell surface

• Oligomerization of its transmembrane domain
  results in
• ion channel activity
• Similar to influenza virus M2 protein, an ion
  channel
• protein that modulates the pH in the Golgi
  compartment
• Ion channel activity of Vpu may be required for
  proper virion
• maturation and assembly by protecting newly
  formed Env protein
• from premature conformational changes in the
  secretory pathway

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Vif Viral infectivity factor, required for
robust replication only in certain cells
Hut78, H9, 1º PBLs
C8166, 293T, HeLa
Permissive Non-permissive
HIV-1 (???
replication
replication
HIV-1 (Dvif)
replication
no replication
Two hypotheses

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

See Simon et. al., Nature Med. 4 1397
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Permissive
Non-permissive
Non-permissive inhibitory cellular factor
overcomed by vif Permissive compensatory factor
similar to vif
Infectivity
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Denv vs Denv/Dvif
Non-Permissive
Permissive
Permissive vs Non-Permissive T Cell Line
Two hypotheses

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

Permissive
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Heterokaryon
Permissive
Non-permissive
wt
Dvif
Dvif
wt
wt
Dvif


Non-permissive inhibitory cellular factor
overcomed by vif
Infectivity
26
(No Transcript)
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Vpr Two Independent Functions

• G2 Arrest
• LTR transcription, i.e.,virus production is more
  efficient during G2
• Augments Nuclear Import of Pre-Integration
  Complex
• Extracellular vpr (from decaying virions, or
  cytosolic leakage from infected apoptotic cells)
  re-capitulates intracellular vpr function
• Induces cell cycle arrest
• Activates HIV replication in latently infected
  cells
• Increased HIV replication in macrophages
• Apoptosis bystander cell killing in lymphoid
  organs and brain

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Vpr G2 Cell Cycle Arrest

• G2 Arrest
• LTR transcription is more active, i.e.,
• virus production is more
• efficient during G2

G0
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Vpr Augments Nuclear Import of Pre-Integration
Complex
Vpr lacks classical NLS sequences --but binds to
Importin-a????????
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Vpr Augments Nuclear Import of Pre-Integration
Complex
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Transport of PICs containing Vpr-GFP fusion
protein
Redanti-tubulin antibodies
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Blueanti-tubulin GreenVpr-GFP RedAlexa-dUTP
PIC/RTC
Nuclear Import of PIC stalled by (a)Anti-dynein
Mab (b)Nicodazole treatment (nicodazole
disrupts microtubles)