1. HIV Cellular Pathogenesis II
Benhur Lee, M.D.

2. 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

3. 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

4. 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:
100%
99%
100%
~50%

5. 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

6. 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

7. 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
NBP-1

8. 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
CTL
HIV
MHC Class I
HIV antigen
51Cr

9. 100% HIV Dnef HIV wt % Lysis E (Effector Cell) CTL 0% 1:2 1:5 1:10
1:20
MHC Class I
HIV antigen
E:T ratio
T (Target Cell)

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

11. 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)

12. 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

13. 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

14. Vif: Viral infectivity factor, required for robust
replication only in certain cells
C8166, 293T,
HeLa
Hut78, H9,
1º PBLs
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

15. Heterokaryon Non-permissive Permissive wt Dvif wt Dvif wt Dvif ++ - ++
Which phenotype will dominate?
Non-permissive
Permissive wt
Dvif wt
Dvif ++ - ++
Non-permissive: inhibitory cellular
factor overcomed by vif
Permissive: compensatory factor
similar to vif
Infectivity

16. Denv vs Denv/Dvif Non-Permissive Permissive Two hypotheses: Permissive
(ensures that
all viruses are
produced from
Heterokaryons)
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

17. Heterokaryon Non-permissive Permissive wt Dvif wt Dvif wt Dvif ++ - ++
Non-permissive: inhibitory cellular
factor overcomed by vif
Infectivity

18. CEM-SS (permissive cells) renders it non-permissive
Expression of CEM15 in
CEM-SS (permissive cells)
renders it non-permissive
After subtractive-PCR for differentially expressed genes

19. 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 (Py-C-C)
Degradation: uracil in DNA recognized
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

20. 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