Transcript HIV Attachment & Entry: Insights into pathogenesis and

HIV
Cellular Pathogenesis II
Benhur Lee, M.D.
HIV Accessory Genes:
Tat
Rev
Essential in vitro and in vivo
Vif
Essential in certain cell types
(Permissive vs Non-permissive cells)
Vpr
Vpu
Nef
Non-essential in vitro, but leads to
attenuated phenotype in vivo
Tat: Transactivator of HIV’s LTR Promoter
– 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
2º structure of HIV
TAR sequence
“bulge”
• Experimental Observations:
“loop”
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
Tat: Transactivator of
HIV’s 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
Rev
• Essential for nuclear export of unspliced
or single spliced viral transcripts
Ran-GTP
RanGAP
Ran-GDP
Rev
Ran
GDP
Importin-b
Importin-b
Arg Rich Domain (ARD)
--binds to Importin-b for
nuclear import
Ran
GTP
Rev
Ran-GTP
Rcc1
Ran-GDP
Rev
Nuclear Export Signal (NES),
leucine-rich domain, binds Exportin-1 (XPO)
cooperatively with Ran-GTP
Importin-b
After nuclear import,
Ran-GDP is converted
to Ran-GTP, and
importin- b dissociates
from Rev
“Free” ARD now
can bind to RRE, but
only in context of
Rev multimers
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 SCIDhu 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)
MGxxx(S)(K)(K/R)
HIV sequence
Conservation in
99%
100%
Nef protein:
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
degradation
Expression of nef alone in T-cell lines can lead to
CD4 downregulation (as determined by FACS)
CD4
..
....................
..................................... ...
........ ......................
...............................
. .............
...............
. .....
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-downmodulation 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
NBP-1
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 HIVinfected cells to CTL mediated lysis in vitro
selectively down-regulates only HLA-A and HLAB, 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
CTL
HIV
HIV antigen
MHC Class I
51Cr
100%
HIV Dnef
HIV wt
% Lysis
E (Effector Cell)
CTL
0%
1:2
1:5
1:10
E:T ratio
1:20
HIV antigen
MHC Class I
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
(ng/ml)
Adv-GFP supnt
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
Vpu: CD4 down-modulation
16 kDa, membrane spanning
Binds CD4 tail in the ER
Targets CD4 for proteolysis via
ubiquitin-proteasome pathway
Vpu mediated CD4 degradation
via ubiquitin-proteasome pathway
Evidence:
Vpu activity disrupted by
inhibitors of proteasomemediated 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
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
Vif: Viral infectivity factor, required for robust
replication only in certain cells
HIV-1 (

HIV-1 (Dvif)
Hut78, H9,
1º PBLs
C8166, 293T,
HeLa
Permissive
Non-permissive
+++ replication
+++ replication
+++ 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
wt
Dvif
Permissive
wt
Dvif
Heterokaryon
wt
Dvif
Which phenotype will dominate?
Non-permissive: inhibitory cellular
factor overcomed by vif
++
-
++
Infectivity
++
Permissive: compensatory factor
similar to vif
Denv vs Denv/Dvif
Non-Permissive
Permissive vs Non-Permissive
T Cell Line
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.
Permissive
Permissive
Heterokaryon
Non-permissive
wt
++
Dvif
-
Permissive
wt
++
Infectivity
wt
Dvif
++
-
Dvif
++
Non-permissive: inhibitory cellular
factor overcomed by vif
Expression of CEM15 in
CEM-SS (permissive cells)
renders it non-permissive
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
Vpr: G2 Cell Cycle Arrest
“Quality Control” phase
Mitosis is not initiated until
DNA is free of damage
G2
G2 Arrest
LTR transcription is more active, i.e.,
virus production is more
efficient during G2
S
DNA
Synthesis
(Mitosis) M
G0
G2 Arrest
nuclear herniation due
to disruption of nuclear
lamin structure
mixing of segregated cell
cycle regulators may lead
to G2 arrest
G1
G2
+
Vpr
PO4-
-
M
Wee1-GFP
Cyc B1-RFP
Vpr:
Augments Nuclear
Import of
Pre-Integration Complex
Vpr=Importin-b analog?
Vpr lacks classical NLS sequences
--but binds to Importin-a
Vpr:
Augments Nuclear Import of
Pre-Integration Complex
Importin-a
Importin-a
Transport of PICs containing Vpr-GFP fusion protein
Red=anti-tubulin antibodies
PIC/RTC
Blue=anti-tubulin
Green=Vpr-GFP
Red=Alexa-dUTP
Nuclear Import of PIC
stalled by:
(a)Anti-dynein Mab
(b)Nicodazole treatment
(nicodazole disrupts
microtubles)