Indian J Med Res 121, April 2005, pp 315-332
315
Biology of the HIV Nef protein
Suman Ranjan Das & Shahid Jameel
Virology Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, India
Accepted March 14, 2005
The accessory Nef protein is expressed by all primate lentiviruses – HIV-1, HIV-2 and simian
immune deficiency virus (SIV). Its expression in the early stages of the viral life cycle ensures
two basic attributes of HIV infection. These are T-cell activation and the establishment of a
persistent state of infection. Nef has a positive effect on viral infection and replication by
promoting the survival of infected cells. Its role in HIV persistence is based largely on the
ability of Nef to downmodulate the surface levels of important molecules at the immune
synapse. These include major histocompatibility complex-I (MHC I) and (MHC II) present on
antigen-presenting cells (APCs) and target cells, and CD4 and CD28 present on helper T cells.
In this review we present these biological properties of Nef from a mechanistic point of view,
and relate them to the structural attributes and interactions of the Nef protein. A brief outline
of the limited studies on Nef from Indian subtype C HIV-1 isolates is also presented.
Key words Apoptosis - HIV-1 - immune synapse - Nef - signaling - T-cell activation
Besides the three prototypical retroviral proteins
(Gag, Pol and Env) and two regulatory proteins (Tat,
Rev) that are essential for viral replication, HIV (and
simian immunodeficiency virus, SIV) also encodes
four so-called accessory proteins called Nef, Vif, Vpr
and Vpu. While these accessory proteins are
dispensable for viral replication in vitro, they perform
essential functions during the viral life cycle in the
host. In vivo studies or those in primary cell types
susceptible to HIV infection have demonstrated that
the accessory gene products can dramatically alter
the course and severity of viral infection, replication
and disease progression. The  nef gene is highly
conserved in all primate lentiviruses  e.g. HIV-1,
HIV-2 and SIV and the encoded Nef protein appears
to be a virulence factor critical for the development
of AIDS 1. It was first identified as an open reading
frame (ORF) that partially overlaps with the 3’-long
terminal repeat of the HIV-1 and was reported to
have a negative effect on viral replication, hence the
name ‘negative factor’ or nef 2-4. Since then,
extensive studies have shown this to be a misnomer
and have characterized multiple cellular pathways that
are regulated by expression of the Nef protein.
Here we discuss the biology of Nef and its various
effects on the host cell in a bid to promote viral
replication and persistence.
Nef as a positive viral factor
Nef is expressed in abundance during the early
phase of HIV infection together with Tat and Rev.
Its mRNA is estimated to represent three quarters
of the early viral mRNA load of the cell 5, 6. Nef
induces high viral titres both in cell culture and
in vivo. Studies in Rhesus monkeys have clearly
demonstrated that an intact nef gene is critical for
attaining high virus loads and development of an
acquired immunodeficiency syndrome (AIDS)-like
316
illness in animals infected with the SIV 7, 8. A similar
requirement for the maintenance of high virus loads
has been demonstrated for HIV-1 in the SCID-Hu
model 9-12. In this system, viruses with an intact nef
ORF replicate faster, achieve higher titres, and deplete
thymocytes better than their nef-deleted
counterparts 13. Moreover, long-term survivors of HIV
infection who show lack of disease progression are
commonly associated with either a deletion in the  nef
gene or defective nef alleles 14-19. In transgenic mouse
models, Nef expression in CD4 positive cells caused
an AIDS like disease 20-23. Of note, the Nef protein
of SIV and HIV are functionally interchangeable 24.
Enhanced virus replication and infectivity are
associated with nef expression upon propagation of
HIV-1 in peripheral blood mononuclear cells (PBMC)
cultures in vitro 25, 26. Increased inocula of nef-deleted
viruses can overcome the decreased infectivity of the
viral progeny but still does not induce illness in infected
animals, suggesting a role for Nef in HIV pathogenesis
as well. The importance of Nef has also been
demonstrated in the Rhesus monkey model of SIV-
induced AIDS, where functional Nef expression is
required for greater infectivity, higher viral loads and
faster disease progression 28.
Nef was originally characterized as a negative
regulator of HIV infection and was thus named as
‘negative factor’2-4. In earlier studies, it was found
that nef-defective viruses replicate slightly faster than
the wild type viruses in CD4+ cell lines2. In addition,
it was shown that Nef could downregulate HIV-1
LTR activity 3. However, these initial findings were
later refuted by other groups 4. In most cases, Nef
either has no effect or a positive effect on viral
replication in vitro. Further, it has also been shown
that Nef does not inhibit transcription of the HIV-1
LTR in a variety of cell types.
The Nef-mediated enhancement of viral
replication is a system-dependent process. Nef
generally has no effect on viral replication in activated
peripheral blood mononuclear cells (PBMC),
activated CD4+ T cells and mature dendritic cell-T
cell co-cultures. On the other hand, a significant role
for Nef in viral replication has been found in post-
infection-stimulated PBMCs or lymphoid cultures,
immature dendritic cell-T cell co-cultures and in the
ex vivo tonsil culture system.
Effect of Nef on virion infectivity and replication
The nef gene of HIV is critical for pathogenesis
and development of AIDS in humans as well as in
animal models. Using the Hela-CD4-LTR- ? -
galactosidase indicator cell line, nef+ HIV-1 was found
to productively infect 5 to 20-fold more cells than
equal amounts of  nef-defective  HIV-129. The
infectivity of nef defective HIV-1 can be rescued by
expressing Nef in trans in the virus-producing cell.
The replication rates of  nef+  and  nef-defective
HIV-1 clones in activated primary blood lymphocytes
(PBL) indicated that Nef directly promotes HIV-1
replication by enhancing the infectivity of virions 30.
Viruses produced from proviral DNAs mutated in nef
are 4 to 40 times less infectious than the wild type
virus in single-round infection assays. The Nef effect
is dependent on the association of this protein with
the plasma membrane and is determined at the stage
of virus particle formation 31.
The inclusion of Nef in the virion may facilitate
the incorporation of Nef-associated cellular kinases
that phosphorylate various substrates, including the
viral matrix protein, important for the production of
fully infectious viral particles 32. Productive HIV-1
infection is also regulated by the ability of Nef to
induce the release of a lymphocyte-stimulating factor
by macrophages. This leads to an environment in
which Nef promotes viral replication in the host by
increasing the pool of substrate lymphocytes without
additional stimuli 33. The effects of Nef on lymphocyte
signaling also involve transcription factors that are
induced in response to signaling from the T cell
receptor (TCR). Manninen et al 34 described a novel
effect of Nef on lymphocyte signaling mediated
independently of the TCR, which results in induction
of nuclear factor of activated T cells (NFAT), a
transcription factor that plays a central role in
co-ordinating T cell activation 34. The effect of Nef
on T cells is mediated by activation of the calcium/
calcineurin pathway and was synergistic with the Ras
pathway in inducing NFAT-dependent gene
expression. Ectopic expression of NFAT in resting
CD4+ T lymphocytes induced a permissive state,
which despite the lack of evidence of T cell activating
effects of NFAT overexpression, supported HIV
replication in these cells in the absence of further
stimulation. HIV replication in these cells is supported
INDIAN J MED RES, APRIL 2005
317
by the overexpression of NFAT target genes IL-2 and
FasL, in the absence of further stimulation 35. The
NFAT protein has also been shown to activate
HIV-1 LTR-directed transcription by interacting with
an unusual binding site that overlaps with the NF-
? B- responsive element. The Nef-mediated
superinduction of IL-2 is a reflection of activation of
both NFAT and NF-? B sites; thus this mechanism
promotes viral replication and spread.
The replication of HIV-1 in vitro is restricted to
dividing (activated) T cells36, 37 and studies detailed
above explain how Nef enhances viral replication by
T cell activation. However, this first requires
expression of Nef in a resting T cell that is refractory
to infection. One mechanism put forth is the
expression of Nef from unintegrated viral DNA 37.
Swingler  et al38 recently proposed an alternative
pathway in which Nef intersects the CD40 ligand-
signaling pathway in macrophages, the first cell type
to be infected by HIV. Physiological stimulation of
CD40 as also Nef expression in macrophages
promotes the release of soluble CD23 (sCD23) and
soluble intercelluler cell adhesion moleculer (sICAM).
These in turn upregulate the expression of co-
stimulatory receptors CD22 and CD58 (by sCD23)
and CD80 (by sICAM) on B lymphocytes, leading to
increased interaction with their corresponding ligands
on T lymphocytes. This causes activation and
proliferation of T lymphocytes, making them
susceptible for HIV infection.
Cell surface expression of critical proteins
The HIV and SIV Nef proteins have been shown
to direct the downmodulation of several critical cell
surface proteins that are a part of the immune
synapse. This is a major viral strategy towards
creating a persistent state of infection. The effects
and their mechanisms are detailed below :
CD4 downmodulation: The CD4 protein is a 55 kDa
type I integral cell surface glycoprotein and a
component of the T cell receptor on MHC class
II-restricted cells such as helper/inducer
T-lymphocytes and cells of the macrophage/monocyte
lineage and serves as the primary cellular receptor
for HIV and SIV 39. The most extensively studied
function of Nef is its ability to dramatically reduce
the steady state levels of CD4 on the cell
surface 40-42. Almost all Nef isolates downmodulate
cell surface CD4 in all mammalian cell types tested,
and under almost all experimental conditions. In
transgenic mice expressing HIV-1 Nef, a significant
decrease in CD4 expression on the surface of
immature double-positive thymocytes is associated
with compromised positive selection of CD4+
lymphocytes 21.  One of the major benefits of Nef-
induced CD4 downregulation could be the
enhancement of viral particle release by preventing
the sequestration of viral envelope by the CD4
receptor or its influence on the assembly and entry
of viral particles 39. The downmodulation of surface
CD4 would also prevent superinfection, an event that
would lead to premature death of the host cell39.
The Env and Vpu proteins of HIV-1 also
downregulate the CD4 receptor 41. However, in
contrast to these two proteins that function at
intracellular sites, Nef acts on CD4 molecules that
have already reached the cell surface. Nef-induced
CD4 downregulation involves accelerating
endocytosis of CD4 followed by its degradation
through the endo-lysosomal pathway 40. It has no
detectable effect on either CD4 synthesis or transport
through the exocytotic machinery 43-45. That Nef
reduces cell surface CD4 through the endo-lysosomal
pathway is supported by the observation that Nef-
expressing cells have more numbers of CD4
containing clathrin–coated pits (CCPs) 46. Further,
lysosomotropic agents block Nef-mediated CD4
downregulation43.
For endocytosis, transmembrane receptors like
CD4 commonly contain specific motifs within their
cytoplasmic tails that serve as a recognition motif for
CCP and that in turn internalize the receptor 1, 47. The
CD4 protein has four hydrophobic amino acids
including two leucine residues in the membrane
proximal region of its cytoplasmic tail that are
necessary for its endocytosis 40, 48. This site is usually
masked by binding of the Src kinase family T-cell-
specific protein kinase, p56 Lck that prevents CD4
internalization mediated by binding to the adaptor
proteins (AP) in the clathrin complex. Nef appears
to bypass the normal routes of CD4 downregulation,
which involves phosphorylation of Ser408 in its
cytoplasmic tail 49, by directly binding the dileucine
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318
motif in the CD4 cytoplasmic region 50. The residues
in Nef involved in contacting CD4 are WL (57-58)
clustered in a proximal flexible loop, and residues G95,
G96, L97, R106 and L110 present in the Nef core
domain 51, 52. The Asp204 residue in Nef is also critical
for CD4 binding as its mutation to lysine (D204K
mutant) completely abolishes binding to CD4 and its
rapid internalization 53. Additionally, Nef itself
contains a dileucine motif, which recruits the clathrin
complex AP-2 subunits via its µ 2 chain 54. Mutations
of the critical Tyr28 and Tyr39 residues in Nef prevent
this interaction, but the D204K mutant still binds to
µ 2 55. Thus, Nef acts as a bridge between CD4 and
µ 2 by displacing p56Lck and its phosphorylation
trigger.
There are four different classes of adaptor
proteins (AP) found in the cell. Of these, AP1 and
AP2 are components of clathrin coats associated with
the trans-golgi network (TGN)/endosomes and the
plasma membrane; AP-3 exists in both clathrin and
nonclathrin coats and is localized to endosomes, while
AP-4 is a part of the non-clathrin coat associated
with the TGN 56.
A recent report using the yeast three-hybrid
method showed that Nef interacts with AP-1 and
AP-3, but not AP-2 and AP-4 57. Stronger interactions
have also been observed between Nef and the
µ  subunits of AP-1 and AP-3, which involves
trafficking between TGN and the endo-lysosomal
compartment 57, 58. These interactions, including
results with CD4-Nef chimeras 59, suggest that Nef
might direct CD4 from the TGN directly to endosomes
without targeting it to the plasma membrane. The
adaptor AP-2 does not interact with Nef and is not a
direct target for regulation by HIV-1. However,
another protein such as the regulatory V1H subunit
of vacuolar ATPase may be responsible for the Nef-
induced removal of CD4 from the cell surface 60-62.
V1H, also called NBP1 or Nef binding protein-1, is a
part of clathrin-coated vesicles and is required for
the acidification of endosomes and lysosomes. Yeast
two-hybrid analysis has identified a diacidic motif in
the C-terminal flexible loop of Nef to be responsible
for binding to V1H 63. The V1H protein binds to Nef
and the µ 2 subunit of AP-2 and strengthens the weak
direct interaction between Nef and AP-2 (Fig. 1).
Another region of Nef, an acidic dipeptide motif,
EE155/156, which was earlier proposed to interact
with  ? -COP (a part of the COP1 coatamer), is
important for CD4 downmodulation 64-66 and was
proposed to target the Nef-CD4 complex from early
to late endosomes and lysosomes (Fig. 1). The
primary isolate Nef233 has an EK motif at this
position (instead of EE), but is fully functional in CD4
downmodulation 67. Thus, Nef mediated  ? -COP
coatamer recruitment is most likely indirect and
depends on a critical cofactor 68. In recent work,
Faure et al 69. have identified the ARF1 GTPase to
be this cofactor responsible for formation of the
transport machinery via Nef binding (Fig. 1).
Nef has also been shown to bind to the novel
human thioesterase II protein and this interaction is
important for CD4 downmodulation70. However, the
Nef protein of the HIV-1 SF2 isolate does not bind to
thioesterase but still downregulates CD4. One of the
Nef residues critical for thioesterase binding, D123,
was also critical for Nef dimerization and was found
to be critical for Nef mediated downregulation of CD4
and major histocompatibility complex I (MHC I)70.
Besides dimerization, N-terminal myristoylation of
Nef leading to its membrane localization is also critical
for Nef-mediated downmodulation of CD4 as well
as the other surface markers71, 72.
CD28 downmodulation: The functional outcome of
TCR engagement is normally decided by the presence
or absence of co-stimulatory signals delivered via
accessory molecules such as CD2873. Ligation of either
TCR or CD28 alone induces minimal levels of T cell
activation, which leads to a state of anergy73. In addition
to surface downmodulation of CD4, Nef also
accelerates internalization of the CD28 co-stimulatory
molecule, which is necessary for maximal T-cell
activation. Genetic and functional studies suggest that
Nef-mediated endocytosis of CD28 is AP-2 dependent
and is mediated by a direct interaction between Nef,
AP-2 and CD28. This activity of Nef is a conserved
phenomenon in multiple isolates of HIV-1 and SIV 74,75.
The LL165/166 and His194 residues of Nef are critical
for CD28 downmodulation74, 75.
The benefit of CD28 surface downmodulation to
the virus is not clear. It has been hypothesized that
by downmodulating CD28, the virus can block T-cell
INDIAN J MED RES, APRIL 2005
319
activation. The interactions between CD28 and B7
molecules and between TCR/CD4 and MHC II
molecules are thought to make critical contributions
to the stability and maintenance of a tight and
prolonged interaction between T cells and antigen
presenting cells (APCs). The Nef-mediated
downmodulation of CD28, in addition to CD4, probably
limits the strong interaction of Nef-expressing T-cells
with APCs. This would enhance subsequent
movement of infected T-cells into the circulation and
to other APCs to accelerate the spread of the virus.
Alternatively, by downmodulating CD28, Nef can
minimize signaling upon engagement of the infected
T-cell with an APC or interfere with normal TCR-
initiated signaling. This effect may be necessary to
prevent activation-induced apoptosis of already
activated and infected cells by further stimulation
through CD3. As Nef activates certain downstream
effectors of signaling pathways such as NFAT-1 and
IL-2, it can replace the normal signaling cascades
that govern antigen-specific T-cell activation and
signaling. It is plausible that the infected cell can still
Fig. 1. Nef-induced downmodulation of CD4 and MHC molecules. Models are presented based on available data on the downmodulation
of surface-expressed CD4, MHCI and MHCII. For CD4 downmodulation, Nef connects the cytoplasmic tail of CD4 with adaptor
protein 2 (AP2) and the V1H vacuolar ATPase, triggering rapid CD4 endocytosis (1). In the early endosome, Nef interacts with CO P1
coatamers through the ADP ribosylation factor 1 (ARF1) and targets CD4 for lysosomal degradation (2). Nef is targeted to the tr ans-
Golgi network (TGN) (3) where it acquires the ability to activate PI3K (4). The resulting formation of phosphatidylinositol-3,4 ,5-
triphosphate (PtdInsP) recruits the guanosine exchange factor ARNO to the plasma membrane leading to the activation of ARF6 (5) .
This accelerates the endocytosis of MHC class I molecules into an ARF6-dependent compartment (6) and subsequently through a
PACS-1/AP1-dependent step into the TGN (7). The MHC class II molecules possibly bind Nef at the plasma membrane and are
relocated to the early endosome (8) enroute to their lysosomal degradation (9). The activation of PI3K and ARF6 are shown by a
change in color from light to dark. Steps for which mechanistic details are not fully available are shown as dash arrows. (conc ept of
figure adapted from Fig.5 of B.M. Peterlin and D. Trono.  Nat Rev Immunol 2003; 3 : 97-107).
DAS & JAMEEL : HIV NEF PROTEIN
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remain activated yet protected from improper
signaling through CD28 downmodulation, thus
disconnecting T-cell activation from antigen
presentation 74, 75.
MHC class I downmodulation: Adaptive immune
responses require recognition of infected cells prior
to their elimination by cytotoxic T lymphocytes
(CTLs). This requires viral peptide presentation
on the surface of infected cells by MHC I
molecules 76-78. To counteract this host defense
mechanism, several persistent viruses such as herpes
viruses, poxviruses and retroviruses, have developed
strategies that target the assembly and trafficking of
newly synthesized MHC I molecules in infected
cells 78. This alters the MHC I presentation of viral
peptides and promotes the escape of virus-infected
cells from CTL surveillance. Notably, the Nef protein
expressed in the early stages of HIV infection carries
out this function. Following infection by HIV/SIV, Nef
downmodulates surface MHC I and this function is
critical for the establishment of infection and
subsequent development of AIDS. The
downmodulation of MHC I protects HIV-infected
cells from CTL-mediated killing and provides a
selective advantage for viral persistence and
replication in vivo 76. Nef selectively downregulates
HLA-A and HLA-B, which present antigens to CTLs
but not HLA-C and HLA-E which helps protect cells
from lysis by natural killer (NK) cells 79-82. A
cytoplasmic tyrosine residue unique to the HLA-A
and HLA-B proteins confers this specificity. However,
it should be noted that Nef does not protect
completely from immune surveillance, as there is a
strong CTL response to HIV infection.
Heterotrimeric functional MHC class I complexes
consists of the MHC I heavy chain noncovalently
attached with ? 2 microglobulin (? 2m) and loaded with
an 8-11 amino acid peptide. The assembly of these
two components and the loading of antigenic peptides
that are generated by the proteosome occur in the
endoplasmic reticulum (ER). Many DNA viruses
such as adeno, herpes and poxviruses interfere with
peptide-MHC I presentation at the level of assembly
of this complex within the ER 83. Nef does not affect
these early processes, but speeds up the internalization
of cell surface MHCI molecules 83-85.
Mutagenesis studies reveal that Nef-mediated
downregulation of CD4 and MHC I molecules by
endocytic trafficking occurs via distinct pathways
(Fig. 1). In the presence of Nef, the synthesis of MHC
I molecule is unaltered and these molecules transport
normally through the ER/Golgi route. However, MHCI
molecules are rapidly internalized from the cell surface
through the endosomes, transported back to
accumulate in the TGN, and misdirected into post-
Golgi clathrin-containing vesicles in Nef expressing
cells 74, 75. Inhibitors of phosphatidylinositol 3-kinase
(PI3K) block this effect 86. The phosphofurin acidic
cluster-sorting signal 1 (PACS1) protein is required
for Nef-mediated downregulation of MHC I to the
TGN by binding to the Nef acidic cluster EEEE
(amino acids 62-65) 87. While PI3K is important for
TGN-derived clathrin-coated vesicles, PACS1 is
required for early endosome to Golgi trafficking, as
in the case of furin-mannose-6 phosphate receptor
(M6PR) transport 88.  The interaction of Nef with
MHC I is transient 89 and two additional Nef sorting
motifs are critically important for MHC I
downregulation. These include the Met20 residue
within the amphipathic helix and the PXXP motif-
containing SH3-domain binding site 51, 86, 90.
Dominant-negative proteins and chemical
inhibitors have been used to explain the sequential
roles of the three Nef sorting motifs 86. MHC I
downregulation is initiated by the binding of the Nef
acidic cluster (EEEE; 62-65) to PACS-1/AP-1. This
is a crucial step to the subsequent PXXP (residues
72-75) mediated ADP ribosylation factor 6 (ARF6)
activation 86, 91. The ARF6 protein connects vesicles
trafficking with actin cytoskeletal rearrangement, and
this endocytosis and recycling of proteins through the
ARF6-dependent endosomal compartment is
regulated by the GTP/GDP bound state of ARF6. Nef
activates the ARF6 GTPase through PI3K and the
guanosine-exchange factor (GEF) ARNO, thereby
triggering the clathrin-independent transport of MHCI
molecule from the cell surface to the TGN through
an ARF6 compartment 86.  It has also been proposed
that the apparent ARF6 dependence of Nef-mediated
MHC I downmodulation could be due to nonspecific
perturbations in membrane trafficking92, 93. The steps
in MHC I downmodulation by Nef are illustrated in
Fig.1.
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321
MHC Class II downmodulation : The  MHC II
protein is expressed in all APCs and present antigenic
peptides to CD4+ T cells94. Thus, it is present on all
cell types that are infected by HIV. Further, human
macrophages accumulate HIV-1 particles in MHC
II compartments95 and MHC II can be incorporated
in the virion96. In chronically infected monocytes,
MHC II antigen presentation is hampered 97, and
HIV-1 Nef is reported to affect the surface
localization of MHC II proteins, reducing presentation
of exogenous peptides to CD4+ cells98-100.
The presentation of antigenic peptides in the
context of MHC II is central to the adaptive immune
system whereby peptides generated from exogenous
sources are presented to T-helper cells.
The Nef protein disrupts MHC II antigen
presentation by two distinct mechanisms. These
include downregulating surface expression of mature
MHC II and upregulating surface expression of MHC
II associated invariant chain (li, CD74). Higher
amounts of Nef expression are required for mature
MHC II downmodulation compared to upregulation
of Ii. Nef-alleles from primary HIV-1 cause surface
upregulation of Ii, a function that is genetically
separable from MHC II surface downmodulation100.
For example, the acidic domain of Nef is involved in
downregulation of MHC II but is dispensable for Ii
upregulation. Mutations in the C-proximal flexible loop
consistently abolish the ability of Nef to modulate Ii
surface expression but had little effect on
downregulation of MHC II. The dileucine motif
LL165/166 and the acidic motif EDE174-176 are
critical for Nef-induced downregulation of CD4 and
upregulation of Ii. Mutations in Pro75 and Pro78 block
MHC I and MHC II downmodulation but have no
effect on Ii upregulation100, suggesting that an intact
SH3-binding PxxP motif is not required for Ii
upregulation but is required for MHC II modulation98-
100. These results illustrate the mechanistic details of
MHC II downmodulation by Nef and emphasize the
importance of this function in viral immune evasion
through inhibition of virus-specific T cell help.
T-cell receptor and cellular signaling pathways
Stimulation of the TCR on mature T cells induces
a series of biochemical events, which eventually result
in the induction of gene expression in the activated
T cell. The initiating signals are activated protein
tyrosine kinases, including members of the Src, Syk/
Zap-70 and Tec families. These kinases phosphorylate
their target substrates resulting in the modification or
metabolism of membrane phospholipids mediated by
enzymes such as phosphatidylinositol 3-kinase
(PI3K), phopholipase C (PLC) and calcium
homeostasis. This leads to the production of second
messengers such as diacylglycerol and inositol
triphosphate, and the activation of small GTP-binding
proteins, such as p21Ras. The ultimate outcome of this
complex intracellular signaling is the activation of
transcription factors leading to gene expression101.
The culture of macaque peripheral blood
lymphocytes7 and a herpes virus samiri-infected
macaque T cell line require the addition of IL-2.
Unlike lymphocytes, this cell line becomes IL-2
independent when infected by nef+ SIV but not by
nef-defective SIV102, suggesting that Nef can play a
direct and positive role in T cell activation. Microarray
analyses have also shown that the total expression
profiles of TCR-activated T cells and Nef expressing
cells are 97 per cent similar103. Nef might initiate the
activation process by myristoylation-dependent
translocation to the plasma membrane and its
interaction with a variety of signaling proteins 104.
Many of these signaling proteins, like Lck or linker
for activation of T cells (LAT), are at least partially
present in glycolipid-enriched microdomains (lipid
rafts) where Nef is also found105. Aggregation of lipid
rafts initiates T cell signaling, similar to the manner
of receptor stimulation by ligands. By binding to
molecules of different compartments, including rafts
and possibly by forming oligomers, Nef may function
as an intracellular cross-linker or adaptor.
The Nef protein lacks any catalytic activity and
acts by influencing kinases or other signaling
pathways within the host cell 106. The strong
conservation of proline-rich motifs among various Nef
alleles and SH3-dependent activation of Src family
tyrosine kinases may be a general property of primate
lentiviruses. The interaction of Nef with the Hck SH3
domain has been shown to be the tightest SH3
domain-ligand interaction so far reported, with a
dissociation constant KD of 0.2 µ M107. Stimulation of
the Hck tyrosine kinase activity by Nef depicts a
DAS & JAMEEL : HIV NEF PROTEIN
322
particularly extreme phenotype resulting in malignant
transformation of Rat2 fibroblasts108. Hck is rapidly
induced following macrophage activation and has
been implicated in multiple signaling events including
phagocytosis, Fc receptor signal transduction, integrin
signaling, and tumour necrosis factor release109.
Two phosphoproteins with molecular weights of
62,000 and 72,000, called Nef-associated kinases
(NAKs), were found to co-immunoprecipitate with
Nef110. One of these proteins is the p21-activated
kinase 2 (PAK2) 111, that has direct effects on
cytoskeletal morphology and apoptotic signaling. Nef
may modulate the effects of PAK2 in mediating
signaling from the cytoplasm into the nucleus and/or
in inducing reorganization of the actin cytoskeleton,
both of which could facilitate steps such as
transcription, reverse transcription or budding.
The enzyme PI3K consists of a regulatory and a
catalytic subunit. Following the generation of
phosphatidylinositol (PtdIns)-3,4,5-Pi, a variety of
proteins are recruited to different membranes via their
lipid binding domains. Among them are the exchange
factors for small GTPases of the Rho, ARF family
and the PDK-1 families112.  The relocalization of
these proteins initiates a broad range of cellular
effects such as proliferation, membrane ruffling,
prevention of apoptosis, and certain vesicular transport
functions. Nef binds the regulatory p85?  subunit of
PI3K. It is possible that Nef assembles PI3K, Vav,
and PAK into a signaling complex, which is required
for increased viral production in a Nef-dependent
manner113.
The Nef protein has also been reported to bind
the c-Raf 1 kinase, which plays an important role in
co-ordinating the Ras-Raf-MAPK pathway114. This
particular binding is dependent on a conserved
DDPxxE174 motif in Nef, which resembles an acidic
consensus Raf-binding motif previously characterized
in p21Ras. Nef is also reported to bind to the 80-kDa
theta isoform of protein kinase C (PKC ? ); this
interaction is reported to modulate PKC activity, since
its normal relocation to the particulate cellular fraction
was inhibited in Jurkat cells expressing Nef115. Nef
also associates with the receptor for activated C
kinase 1 (Rac1), a known intracellular receptor for
PKCs116. Rac1 is in equilibrium between a cytosolic
and membrane bound state, partially colocalizes with
Nef, and enhances its in vitro phosphorylation by
PKCs117.
The expression of Nef in T cells unleashes a
series of signaling events that are similar to those
that occur following T-cell activation through the TCR
and co-stimulatory pathways. This results from the
ability of myristoylated Nef protein to localize at the
plasma membrane and assemble multiprotein signaling
complexes through its various interacting domains.
Nef and apoptosis
HIV induces apoptosis in both infected and
uninfected immune effector cells118. Nef induces the
expression of both Fas (CD95) and the Fas ligand
(CD95L) in infected cells119, 120, with CD95L aiding
immune evasion by inducing the apoptosis of HIV-
specific cytotoxic T-cells (CTLs) 119.  The apoptosis
signal regulating kinase 1 (ASK1) is a key signaling
intermediate in the Fas and TNF-?  death signaling
pathways and was reported to bind Nef 121. This
association is reported to result in the inhibition of
ASK1 kinase activity (Fig. 2) and the downstream
induction of c-Jun N-terminal kinase (JNK) and
apoptosis122. The binding of Nef to ASK1 is lost on
disruption of its N-terminal myristoylation site as well
as by a R106A mutation, which is implicated in the
binding of a p21–activated kinase (PAK) family
protein123.
The association of Nef with ASK1 reveals a
mechanism by which HIV-1 Nef can alter the
intracellular milieu of virally infected host cells by
enhancing their resistance to Fas and TNF?  mediated
apoptosis (Fig. 2). The Nef protein also represses
death signaling by Bad, a pro-apoptotic member of
the Bcl-2 protein family whose expression is induced
by HIV, and that triggers apoptosis at the level of
mitochondria123. The Nef-mediated activation of
PI3K and PAK results in the phosphorylation of Bad
resulting in release of the anti-apoptotic Bcl-X L
protein from a Bad/Bcl-XL complex and enhancement
of cell survival and virus production124. Through its
N-terminus (residues 1-57) Nef also interacts with
the p53 tumour suppressor protein. This interaction
results in the destabilization of p53, thereby decreasing
its pro-apoptotic, transcriptional and DNA-binding
INDIAN J MED RES, APRIL 2005
323
activities, and protecting HIV-1 infected cells from
p53-mediated apoptosis125. Thus Nef protects the
infected cell through two mechanisms. It blocks
external death signals coming from CTLs or auto-
reactive FasL through the inhibition of Ask1. Internal
death signals that are generated on account of
disturbed cellular homeostasis are inhibited through
the phosphorylation of Bad. The inhibition of p53
further enhances the anti-apoptotic effects of Nef in
the infected cell (Fig. 2).
Nef genes from Indian isolates of HIV-1
While most of the functions of Nef are conserved
across the different HIV-1 subtypes, studies on Nef
functions have largely been carried out with the
protein from subtype B isolates, mainly the NL4-3
isolate. We have earlier reported the cloning,
sequencing and phylogenetic analysis of nef genes
from primary HIV-1 subtype C isolates from India126.
Our results also showed that in addition to env and
gag,  nef sequences could also be used to reliably
subtype isolates belonging to the M group of HIV-1.
These results have recently been confirmed by
characterizing more nef sequences from Indian
seropositive cases127.
Besides full-length nef genes, we also cloned
truncated forms with major in-frame deletions from
the primary Indian isolates126. Recently, we have
studied the functional aspects of three of these nef
alleles, F2 expressing the full-length protein, and D1
and C2 expressing proteins with in-frame deletions
of critical motifs (Fig. 3A). While the F2-Nef protein
was competent in downregulating cell surface CD4,
MHC I and MHC II, the D1-Nef protein
Fig. 2. Nef and apoptosis. Nef stimulates the expression of Fas Ligand (FasL) on the surface of HIV-infected cells through an as yet
poorly characterized pathway (1). This leads to increased killing of bystander cells expressing the Fas receptor (Fas). When HI V-
specific CTLs come into contact with their targets, the latter are killed through the Fas-FasL pathway. In infected cells Nef b locks this,
and the TNF receptor-mediated death pathway, by inhibiting the apoptosis-associated kinase 1 (Ask1) (2). Nef directly binds p53  and
prevents the induction of p53-mediated apoptosis in HIV-infected cells (3). It also unleashes the anti-apoptotic effects of Bcl -2 and
Bcl-XL by PAK-mediated phosphorylation of the pro-apoptotic molecule BAD (4) causing its release from a Bcl-2 or Bcl-XL
complex (5). The free Bcl-2 and Bcl-XL block cytochrome c release from the mitochondria (6). This pathway is similar to that in itiated
by Akt-mediated phosphorylation of BAD following cytokine binding to its cognate receptor. Full and dotted lines indicate the d irect
and indirect effects of Nef, respectively. (concept of figure adapted from Fig.6 of B.M. Peterlin and D. Trono.  Nat Rev Immunol
2003; 3 : 97-107).
DAS & JAMEEL : HIV NEF PROTEIN
324
downregulated MHC II, but not CD4 or MHC I (S.R.
Das; unpublished results). The C2-Nef protein has a
major deletion that encompasses the PACS-1, SH3
and PAK1/2 binding sites, and was unable to
downregulate any of these surface proteins (S.R. Das;
unpublished results). Surprisingly, the WL motif
required for binding to CD4 was preserved in C2-
Nef but it was still insufficient to downregulate cell
surface CD4, suggesting that flanking sequences
might also be critical. The C2-Nef protein is 121 amino
acids long and is missing a large part of the core
region. It is possible that this allele is non-functional
and is compensated by other functional alleles in the
same patient. However, its selection in a rapidly
evolving viral quasi-species population is intriguing.
Structure-function relationship of the Nef protein
The variety of functions carried out by Nef is
based on its ability to interact with multiple cellular
proteins. These proteins and their interacting sequence
motifs in Nef are shown in the Table. The HIV-2 and
SIV Nef proteins are 10-30 amino acids longer
compared to that of HIV-1104. While it has not been
possible to crystallize the full-length Nef protein,
structural information has been put together based
on the X-ray structure of its core domain and solution
nuclear magnetic resonance (NMR) structures of its
N- and C- terminal domains. The emerging picture
shows a flexible N-terminal of about 70 residues
followed by a well-conserved and folded core domain
of about 120 amino acids. The highly conserved
MGGxxS sequence at the N-terminus of Nef is
myristoylated at the G2 position; this modification is
critical for its attachment to the inner face of the
plasma membrane and is required for all the functions
associated with Nef. The G2A mutation has been
shown to abrogate the CD4, MHC I and MHC II
downmodulation properties of Nef.  A G2A mutation
in the F2-Nef protein from Indian subtype C HIV-1
also showed similar effects (S.R. Das; unpublished
results).
The solution structure of Nef either alone or
bound to a peptide from the cytoplasmic tail of CD4
has been determined by NMR51. The NMR structure
of a peptide shows that the N-terminal domain of Nef
forms a well-ordered alpha helix from residues 6 to
22. The core domain is the only part of the Nef
protein, which has a stable tertiary structure. This
fragment has been resolved by NMR 51 and
crystallography128, 129. It forms an ? -?  domain in
which a central anti-parallel ?  sheet of four strands
(? A-? D) is flanked N-terminally by two long anti-
parallel ?  helices (? A and ? B) and C-terminally by
two short ?  helices (? C and ? D). A proline-rich
sequence P69 to P78 forms a type II polyproline helix
(aa 70-77), which represents the main binding site
for Src family kinases through their SH3 domains.
This domain is followed by two ?  helices (aa 81-120),
a four-stranded anti-parallel ? -sheet (aa 121-186), and
two additional ?  helices (aa 187-203) 104. Residues
60-71 and 149-180 form flexible solvent exposed
loops. The three proximal helices of the Nef core
domain (aa 70-120) could theoretically form a cavity
accessible to drugs, which could disrupt interactions
between Nef and Src family of kinases.
The motifs critical for Nef function and its binding
partner are summarized in the Table. Most of the
motifs that are critical for binding to different cellular
proteins are in the core domain and are exposed for
easy accessibility to their binding partners. A space-
fill model of the core region of Nef (Fig. 3B) shows a
large binding surface on one side of the protein that
is responsible for MHC I downmodulation through
the binding of PACS-1 and SH3 domain-containing
proteins. The thioesterase-binding site or the
dimerization interface is exposed as seen in view 2
(Fig. 3B); the homodimerization of Nef is stabilized
by a salt-bridge between residues D123 (within the
FPD motif) and R105 that forms the PAK1/2 binding
site (view 1; Fig. 3B). As these residues appear to
be present on different surfaces of the Nef protein,
the homodimer is likely to be asymmetric. However,
dimerization per se is likely to increase the collection
of cellular proteins in a Nef-mediated complex. In
the absence of a clear biochemical function, for
example an enzymatic activity, associated with it, Nef
acts as an adaptor protein and may provide a surface
for bringing together critical signaling molecules within
the infected cell.
Conclusion
A state of persistence favours the life cycle of
HIV. Being a retrovirus, its genome is integrated into
the host genome; therefore, it makes sense for HIV
INDIAN J MED RES, APRIL 2005
325
Table. Cellular proteins interacting with Nef
Name Region of Nef critical Region of the cellular
for binding/function partner critical for binding
Protein modification:
N-myristoyl transferase MGxxxS1 Unidentified 71, 72
HIV-1 protease CAW? LEA55 Unidentified  71, 104
Signalling:
Src family tyrosine kinases42,71,104,128,129
Hck PxxPxR SH3 domain 42, 107, 109
Lck PxxPxR, N-terminal SH3 domain 104, 128, 129
Fyn PxxPxR SH3 domain 104, 128, 129
Lyn PxxPxR SH3 domain 104, 128, 129
Vav PxxPxR SH3 domain 104, 128, 129
Src PxxPxR SH3 domain 104, 128, 129
Others
PAK1/2 (NAK) PxxPxR, MGxxxS, RR105, Unidentified  111, 113, 124
Leu112, FPD121
MAPK (Erk1) PxxPxR SH3 domain 104, 128, 129
c-Raf1 kinase DDPxxE174 Unidentified 114
PI3K N- and C-terminal of Nef p85  subunit  42, 124
TcR? PxxPxR Unidentified  42, 125
p53 N-terminal1-57 Unidentified 121
Ask1 Unidentified Unidentified 121
Cell surface receptors:
CD4 MGxxxS1, WL57, L110, FPD121 Cytoplasmic tail 39-71
D/ExxxLL165, EE154, DD174
CD28 MGxxxS1, Unidentified  73-75
MHCI MGxxxS1, M20, EEEE62, PxxPxR, Cytoplasmic tail 71,76-93
FPD121, EE154
MHCII MGxxxS1, D/ExxxLL165, EEEE62, Unidentified  894-100
PxxPxR,
Trafficking:
PACS-1 EEEE62 Unidentified  86-88, 71
Human thioesterase (p35) FPD121 Unidentified 70-71
? -COP EE154 Unidentified  65, 66, 68, 71
Adaptor proteins AP-1/2/3 D/ExxxLL µ  subunit of
AP1/2/3  55, 57-59, 63, 71
? 1 of AP-1 D/ExxxLL ? 1 of AP1 58, 59, 71
V1H DD174 Unidentified 60-62, 71
326 INDIAN J MED RES, APRIL 2005
to promote the survival of infected cells. However,
continuous expression of viral proteins at high levels
also poses a risk for the infected cell to be recognized
and eliminated by virus-specific CTLs. In addition,
antibodies generated against HIV proteins, especially
the surface proteins, would target extracellular virions,
further limiting the spread of infection. The HIV
counter-attack to the host immune response is to
downregulate those host molecules that are critical
for the development of cellular and humoral immune
responses.
In this strategy, the Nef protein plays a central
role by downmodulating the surface expression of
CD4, MHC I, MHC II and CD28 proteins critical for
the formation of an immune synapse. Lower surface
levels of CD28 in infected T cells ensure that these
cells cannot provide effective help following TCR
engagement. Correspondingly, in professional antigen-
presenting cells such as macrophages and dendritic
cells that are the first cells to be infected by HIV,
downmodulation of surface MHC I and MHC II
ensures poor presentation of HIV peptides to TCRs
on helper or cytotoxic T cells. In addition, Nef-
mediated reduction in MHC I expression on the
surface of HIV-infected cells helps these cells escape
from virus-specific CTLs. The overall effect of this
strategy is to reduce T cell help for generating
effective virus-specific antibody and cytotoxic
responses, as well as reduce the engagement of
infected cells and virus-specific CTLs. The Nef
protein also promotes the survival of infected cells
by inhibiting “outside-in” as well as “inside-in” death
signals; in the latter case, p53-independent as well as
dependent pathways are invoked.
Besides an active role in promoting viral
persistence, Nef initiates a transcriptional programme
in T cells similar to that seen in TCR-activated T cells.
This has an overall effect of enhancing viral replication
and virion infectivity. The Nef protein interacts with
a wide range of cellular proteins, many known to
Fig. 3. Primary sequence and structure of the HIV-1 Nef protein.  (A) The amino acid sequence of a prototype Nef protein from the
HIV-1 subtype B NL4-3 isolate (Nef NL) is shown and aligned to a subtype C Nef consensus sequence (Cons C), and sequences of
full-length (Nef F2) and mutant (Nef D1, Nef C2) Nef proteins from Indian HIV-1 subtype C isolates. Distinct functional regions  of
Nef are highlighted in different colors. (B) A space-fill model of the core region of Nef is shown with distinct functional regions
highlighted in different colors. View 1 shows a frontal representation of a majority of functional domains. View 2 shows a side
representation highlighting the dimerization motif. View 3 is a top-down view highlighting the surface required for MHCI downmo dulation
through PACS-1 and SH3 domain interactions.
327
perform critical functions in signaling pathways.
Structure-function studies on the Nef protein have
identified a number of motifs involved in protein-
protein interactions and favour a model wherein Nef
acts as an adaptor to accumulate signaling complexes
in the infected cell. The downstream effects of these
signaling pathways position Nef as a critical “positive
factor” in HIV pathogenesis.
Acknowledgment
The award of a Senior Research Fellowship of the CSIR
(India) to SRD and an International Senior Research Fellowship
of the Wellcome Trust (UK) to SJ are gratefully acknowledged.
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