Journal of General Virology (I 996), 77, 2379-2392.
Printedin Great Britain
From negative factor to a critical role in virus pathogenesis:
the changing fortunes of Nef
Mark Harris
MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Glasgow G61 IQH, UK
1. Introduction
The net gene is unique to the primate lentiviruses. Over the
last 5 years it has become clear that nefplays a key role in the
pathogenesis of AIDS and this has led to a resurgence in
interest in the function of what was originally considered to be
a 'negative factor', dispensable for virus growth, at least in
vitro. This review is an attempt to summarize our current
knowledge of the biochemistry and cell biology of the Net
protein and to put this into the context of the requirement for
net in vivo.
The open reading flame encoding the Net protein is
situated at the 3' end of the viral genome, overlapping the
3"LTR (see Fig. 1). It is translated from a single coding exon
present on a number of multiply spliced mRNA species. Netspecific mRNA has been reported to constitute up to 80 % of
the viral mRNA present in infected cells at early time points
after infection (Robert-Guroff et al., 1990); however, whether
this equates to a similar preponderance of Net protein product
remains to be elucidated. In human immunodeficiency virus 1
(HIV-1) the open reading flame is 618 bp, coding for a protein
product of 206 amino acids, but there is a degree of length
heterogeneity between isolates. HIV-2 and simian immunodeficiency virus (SIV) Net products are somewhat larger,
between 250 and 265 amino acids. The primary amino acid
sequence of Net is highly variable (Delassus et al., 1991; Harris
et al., 1992; Shugars et al., 1993) and this variability in the
primary structure of Net manifests itself in marked differences
of the mobility of different Net proteins in SDS--PAGE and,
more significantly, has possibly contributed to the contradictory results published about Net function over the last
decade.
2. Structure of Nef
(i) The role of rnyristoylation
Until recently, very little was known about the threedimensional structure of the Net protein. Native Net is
cotranslationally modified by the addition of a myristoyl
Author for correspondence: Mark Harris.
Fax +44 141 330 5602. e-mail [email protected]
0001-4127 © 1996 SGM
group to a conserved N-terminal glycine (Allan et al., 1985).
Myristoylation of Net has been shown to be critical for its
function in a number of assay systems and is required for
association of Net with cytoplasmic membrane structures, as
will be discussed later in this review. However, the role of
myristoylation in determining the tertiary structure of Net has
not been addressed. It is conceivable that, by analogy to other
myristoylated proteins such as the catalytic subunit of cAMPdependent protein kinase (Zheng et al., 1993), the photoreceptor protein recoverin (Tanaka et al., 1995) and the
MARCKS family (Aderem, 1992), the myristoyl group of Net
may play a structural role, possibly by interacting with
hydrophobic regions within the protein itself. Taking these
analogies further provides a plausible explanation for the
observed distribution of myristoylated Net between membrane
and cytosol (see section 3 below): recoverin undergoes a Ca ~+myristoyl switch whereby the myristoyl group in the Ca 2+free protein is sequestered within the protein but is exposed
upon binding of Ca 2+, allowing interaction with the membrane
(Tanaka et al., 1995). MARCKS is released from the membrane
by protein kinase C (PKC)-dependent phosphorylation, which
introduces negative charge to the protein, neutralizing electrostatic interactions with acidic phospholipids (the myristoylelectrostatic switch) (Aderem, 1992). Two conformationally
distinct forms of Net might exist in which the myristoyl group
is either anchored into the lipid bilayer or sequestered by
hydrophobic interactions with the protein itself. This conformational switch could be generated by an additional
modification such as phosphorylation or by interactions with
specific cellular proteins. This hypothesis is indirectly supported by two structural studies of recombinant Net expressed in
Escherichia coil Several years ago a nuclear magnetic resonance
(NMR) study (Freund et at., 1994a) revealed a two-domain
structure: firstly, a compactly folded, hydrophobic core domain
extending from amino acid 66 to the C terminus that was
relatively resistant to proteolysis apart from a predicted loop
between Glu-155 and Glu-172; and secondly, an N-terminal
domain that exhibited no defined structure and was highly
accessible to proteolytic cleavage, suggesting a surface
location. More recently, the solution structure of a partially
deleted Net protein was solved using NMR (Grzesiek et al.,
1996). Again, this study revealed a disordered N-terminal 39
amino acids and a loop between amino acids 159-173. Indeed,
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!37 ~.
rBv
gag
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5 'LTR
/[
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VpU
Fig. 1. Structure of the HIV-I genome. The open reading frame coding for Nef is situated at the 3' end of the genome,
overlapping with the 3'LTR. In the HXB2 genome only one base pair separates the termination codon for gp160 and the Nef
initiation codon. However, in HIV-2 and SIV the Nef open reading frame overlaps with the 3' end of e n v by about 170 bp.
I0
BHI 0
MGGKWSK
HXB2
..........
20
30
S SWGWPAVRERMRRAEP
I..LT
40
50
AADGVGAASRDLEKHGAI
T..A
HXB3
................................................................
MAL
.........
I .... KI...I..
ELI
.........
I .....
I...I..TN
I .....
I...
Z6
...R
SF2
.......
.....
RF
........
BRU
..............
NL43
..........
R.MG..S.I
KMG
I..TD.RRTDP
.........
V ................
........
I .....................
120
IHSQRRQDILDLWI
HXB2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HXB3
. . . . . . . . . . . . . . . . . . . . . . .
W. . . . . . . . . . . . . . . . .
Mu~.L
VW. PK..E
.....
V .........
W ..........
ELI
.W.KK..E
.....
V.N...I...W
.....
V.N...I...W
.W. KK..E
.W .....
100
SH FLKEKGGLEGL
E.SD
E.S
R ...........
G.F
............
......
R ...........
E.L
...............
.......
R ...........
L .................
A.T
.....
ED.D
R .............
.......
R .........
L.I
D. .
.............
F ................
D..
..............................................
A ..............................................
1 SO
160
t 70
180
190
200
YHTQGYF~D*QNYTPGPGIRYPLTFGWCY~LVPVEPEK~EEANKGENTSLLHP~SLHGMDDPEREVLEWRFDSRLAFHHMARELHPEYF~N~*
E ...............
........
. . . . . . . . . .
..........
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140
EEEEVGFPVTPQVPLRPMTYKAAVDL
T ...................
V ....................
130
S.EPP
T ...........
BH10
Z6
RDT
V .................
QK ...................
90
..............................................
ST ..........
T ...................................
110
SF2
.....
.......................
RAEP
80
K ...................................
TP. TETGVG.VSQD.V.Q..D.C..AA..SP
.........
70
TS SNTAANNADCAWLEAQ
...................................
. . . . . . . .
60
V .........
V . . . . . . . . . . . . . . . . .
F .......
...................
..................
D.I
F ....
MS..EV
W ........
F .......
BRU
.......................
NL43
................................
W ........
T .........
E...NC
FE .... D. REV...TE..TNC
F .........
VF..K
....
E .... D.QEV..DTE..TN
W ..................
RF
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V . . . . . . . . . . . . .
R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V .... E..NN
D.V...TE...N
....
I.Q...E.A
.....
ICQ...E
.....
.....
K.K...S..LR.R...Q
.... Q..K...N
CQ...E.T
.....
M .....
.....
IC ........
.....
K...N
E.A.K...V
.....
K...V.K
....
Y.D..
.....
E.K...M...FY..*
.....
E.K...M...
FY.D..
K ..............
.........
V...K
Y.D..
.... Y.D..
V .................
D.V
........................................
V .............
V .................
D.V
........................................
V .............
Fig. 2, Variation in the primary amino acid sequence of HIV-I Nef. An alignment of the amino acid sequences of ten laboratory
isolates of HIV-I Nef, Dots indicate identity with the BHI 0 sequence on the top line of each panel. An asterisk indicates a
termination codon. Two gaps have been introduced to aid the alignment of clones with insertions relative to BH10. It can clearly
be seen that the majority of the variation is concentrated at the N and C termini, flanking a central more highly conserved
region.
the investigators were only able to solve the structure by
deleting these two regions. It is tempting to suggest that
addition of a myristate residue would confer some structural
constraints on the N-terminal domain, particularly if the
myristate were embedded in a binding pocket. In passing, it is
pertinent to note that recombinant Nef produced in E. coli is
efficiently myristoylated in vitro by purified recombinant
human N-myristoyltransferase (NMT; Jeff Mcllhinney, personal communication) suggesting that not only is the
N-terminal sequence of Nef an extremely good substrate for
NMT but that the N terminus of non-myristoylated Nef is
exposed. Intriguingly, the boundary between the two domains
identified in the earlier study coincides with both a highly
conserved run of acidic residues and also a specific cleavage site
for the HIV-1 protease (Freund et al., 1994 b; GaedigkNitschko
et al., I995). Cleavage of membrane-bound Nef by protease
would release the C-terminal domain allowing it to relocate
within the cell and could be potentially important for Nef
'.38(
function. However, cleavage of Nef by protease has yet to be
demonstrated in vivo.
(ii) Does Nef exist in a n oligomeric form?
A number of studies have suggested that Nef is able to
form dimers or higher-order structures both in vitro and in vivo.
Baculovirus- or E. coli-expressed HIV-I Nef formed both
disulphide and non-covalently linked oligomers (Kienzle et aL,
I993). Although the cytoplasm is generally thought to be a
reducing environment there is evidence that the intracellular
reduced glutathione concentration of HIV-infected lymphocytes is low, conditions that might allow the formation of
disulphide bridges. A proposed leucine-repeat motif in HIV-2
Nef was reported to be involved in oligomer formation in vitro
(Hodge et al., 1995), and it was suggested that disulphide
bonds were important both for dimer formation and for the
structure of a Nef monomer as Cys-Gly mutants disrupted
dimer formation even in the absence of reducing agents.
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Furthermore, they demonstrated that oligomeric forms of HIV2 Nef could be immunoprecipitated from infected H9 cells and,
interestingly, only the dimeric form was phosphorylated. We
have consistently observed Nef dimers under both reducing
and non-reducing conditions and also have preliminary
evidence from the yeast two-hybrid system for Nef dimer
formation in vivo. It is clear that the whole question of whether
Nef oligomer formation is physiologically relevant or merely
an artifact of experimental procedure needs to be addressed.
The first step in this process must surely be the precise
definition of amino acid sequences within Nef responsible for
the observed oligomerization. It is still possible that disulphide
bond formation might play a role in vivo; there is evidence
from a number of laboratories that Nef is associated with
intracytoplasmic membrane structures and indeed that Nef is
secreted (Fujii el al., 1993; Otake et al., 1994). If Nef were
present within the lumen of the endoplasmic reticulum (ER)
and/or Golgi apparatus it would be in a non-reducing
environment; alternatively, Nef might be protected from the
reducing environment within the cytoplasm by local conditions.
3. Subcellular localization of Nef
This discussion leads into a consideration of the subcellular
localization of Nef. Nef was originally described as a
cytoplasmic protein (Franchini el al., I986) and it is generally
accepted that the protein is present both as a soluble
component and associated with cytoplasmic membrane structures. Association with the Golgi apparatus (Ovod el al., 1992)
or the nuclear membrane (Kienzle et al., 1992) has been
reported. In transiently transfected Cos cells (Yu & FeMed,
I992; Kaminchik el a]., 1994), stably transfected HeLa cells and
recombinant baculovirus-infected Sf9 insect cells (M. Harris,
unpublished data) membrane association was absolutely dependent on myristoylation. However, studies with other
myristoylated proteins, such as the Src family tyrosine kinases,
have indicated that myristoylation alone is not sufficient to
stably anchor a protein into the membrane (Resh, 1994). The
presence of a polybasic region that interacts with acidic
phospholipids in the inner leaf of the plasma membrane (Zhou
el al., 1994), or an additional acylation event such as
palmitoylation (Resh, 1994), is required for stable interaction
with the membrane. There is a concentration of basic residues
near the N terminus of Nef that could function as a polybasic
region, but there is no evidence for this, nor is there any
evidence for additional acylation of Nef. However, it is
intriguing to note that the C-terminal amino acid of Nef is a
highly conserved cysteine. Although this cysteine is not part
of a classical CAAX box famysylation motif it is conceivable
that it might be a substrate for acylation (especially palmitoylation), given its location at the C terminus.
Nef has also been demonstrated to be present in the nucleus
in chronically infected astrocytes (Kohleisen et al., 1992),
infected T lymphoid cells (Ovod et al., 1992), B cells (Kienzle
el al., 1992) and T cells (Murti et al., 1993) constitutively
expressing Nef and in transiently transfected Cos cells (Yu &
Felsted, 1992). In the case of chronically infected astrocytes
and transiently transfected Cos cells, only non-myristoylated
Nef was localized to the nucleus, suggesting that myristoylation may in some way function to retain Nef in the cytoplasm.
The role of myristoylation may not be simply to anchor Nef
into cytoplasmic membranes as both myristoylated and nonmyristoylated Nef are present as cytosolic proteins. Rather,
myristoylation may influence the structure of Nef as discussed
in section 2(i) above, occluding a genuine or cryptic nuclear
localization signal. Whether nuclear Nef has a functional role in
virus replication remains to be elucidated.
4. Function of Nef: early studies suggested
that Nef was a GTP-binding negative factor _
Some of the earliest assays for Nef function were driven by
the observation of a limited homology between Nef and the
nucleotide-binding sites of G-proteins (Samuel et al., 1987;
Guy et al., i987). Thus, it was demonstrated that E. coil-derived
Nef protein could both bind and hydrolyse GTP and in
addition could autophosphorylate (Guy et al., 1987). However,
since this study, five reports failing to reproduce this data have
been published (Kaminchik et aL, 1990; Backer et aL, 199I;
Nebreda et al., 1991; Matsuura el al., 1991; Harris et aL, 1992).
In retrospect, it seems likely that the results were due to
contamination with a bacterial GTP-binding activity. The
material used in the first study was resolubilized from insoluble
inclusion bodies by SDS treatment and was only 70 % pure.
Subsequent studies used highly purified Nef proteins with a
number of different primary amino acid sequences produced
either in bacteria or in recombinant baculovirus-infected insect
cells. It has to be said that the homology between Nef and Gproteins is not strong: three motifs have been defined that
constitute a GTP-binding site, and both the primary sequence
and the spacing of these motifs are absolutely conserved in all
GTP-binding proteins (Dever el al., 1987). The first motif
(GxxxxGK) has a limited match in Nef (KGGLEGL), but this
does not appear to be a close enough fit to form part of a GTPbinding domain. Likewise, the homologies between Nef and
the other two motifs required for GTP binding (DxxG and
NKxD) are similarly weak.
Early data from a number of laboratories suggested that
Nef repressed virus replication, Indeed, this is where the
acronym 'Nef' arose (NEgative Factor). The majority of these
studies were performed with infectious proviruses containing
deletions or frameshift mutations in the Nef-coding sequence
and showed that these deletions produced virus that replicated
to higher levels than wild4ype virus (Fisher el al., 1986;
Terwilliger el al., 1986; Luciw el a]., 1987). Similar effects were
observed following infection of Nef-expressing cell lines with
Nef-deleted viruses (Cheng Mayer et aI., 1989). It was further
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reported that these effects were a manifestation of the
inhibition of LTR-driven transcription by Nef (Ahmad &
Venkatesan, 1988; Niederman et al., 1989; Maitra et al., 1991).
These observations prompted a number of groups to publish
data demonstrating that such effects could not be confirmed
and indeed that Nef had no effect on virus replication in the in
vitro systems used (Hammes et aI., 1989; Kim et al., 1989). It is
clear now that Nef expression is in fact critical for efficient virus
replication under conditions that more closely mimic the in
vivo environment of virus replication, i.e. low multiplicity
infection of quiescent lymphocytes that are subsequently
activated. These data will be dealt with in greater detail later in
this review.
5. Function of Nef: a positive role in virus
replication
(i) CD4 down-modulation
Although the early work of Guy et al. (1987) did lead a
number of groups up a blind alley, pursuing the GTP-binding
activity of Nef, they may be credited with the initial
observation that Nef expression leads to the down-modulation
of cell-surface CD4 expression. This has now been confirmed
by many groups (Garcia & Miller, 1991; Anderson et al., 1993 ;
Aiken et al., 1994; Rhee & Marsh, 1994a, b; Greenway et al.,
1994; Bandres et al., 1995) and appears to be a highly
conserved function between diverse Nef isolates (e.g. HIV-1
laboratory strains and SIVmac239) (Foster et al., 1994;
Sanfridson et al., 1994). CD4 down-modulation has been
widely accepted as a reliable indication of functionality for a
particular Nef isolate but this is perhaps a rather naive
assumption. Until recently, the only Nef alleles that have failed
to down-modulate CD4 are non-myristoylated mutants and
mutants that produce unstable or poorly expressed protein
products (Mariani & Skowronski, 1993; Aiken et al., 1994).
Recently, mutations of charged residues between amino acids
155 and 175 that significantly impaired CD4 down-modulation
were described (Aiken et al., 1996). In particular, a mutation of
Asp-174/175 gave rise to a protein that was completely
inactive and mutations of Lys-158 and Glu-160 produced a
temperature-sensitive Nef protein that was impaired at 37 °C
but not at 32 °C. Interestingly, these mutations coincide with
the exposed loop identified in structural studies of Nef (see
section 2(i) above) suggesting that this loop might be involved
in binding to CD4 (see below) or interacting with components
of the mammalian endocytic pathway to induce CD4 downmodulation.
The sequences in CD4 that are responsive to Nef-mediated
down-modulation have been studied in detail, but contradictions still exist in the literature. As expected, the cytoplasmic
domain was shown to be critical (Garcia et al., 1993; Anderson
et at., 1994a), in particular a dileucine motif that has been
identified as an endocytosis signal in a number of transmembrane glycoproteins (Letoumeur & Klausner, I992; Aiken
-~38~
et al., 1994). However, there is little consensus concerning the
precise sequence requirements for down-modulation: truncations of the cytoplasmic domain from the C terminus have
been performed by a number of laboratories resulting in the
definition of the minimal Nef-responsive domain as extending
to amino acid 425 (Bandres et al., 1995), 424 (Anderson et al.,
1994a), 418 (Salghetti et al., 1995) or 416 (Aiken et al., 1994).
Two of these studies (Anderson et al., 1994a; Salghetti et al.,
1995) demonstrated that internal deletions either between
residues 397-417 or 403-418 (including the dileucine) were
unresponsive to Nef. Conflicting reports concern the requirement for the two cysteines involved in binding to p56Lck
(residues 420 and 422). These residues were shown to be
required for Nef responsiveness in Jurkat cells (Bandres et al.,
1995) but not in NIH3T3 cells (Anderson et al., 1994a). A
further confusion arises from the fact that different groups used
different nef alleles in their experiments (SF2, NL4-3 or
primary isolates). The biochemical mechanism of downmodulation also remains the subject of much speculation.
Although physiological down-modulation of CD4 (eg by
antibody cross-linking) requires phosphorylation of serine
residues in the cytoplasmic tail by PKC (in particular serines at
408 and 415), these serines were shown to be dispensable for
Nef-mediated down-modulation (Garcia & Miller, 1991). It
seems likely therefore that the latter occurs by a novel
mechanism. Although the rates of CD4 synthesis and transport
to the plasma membrane are unaffected, Nef expression does
appear to induce an increase in the rate of endocytosis of CD4
(Aiken et al., 1994; Rhee & Marsh, 1994b; Sanffidson et al.,
1994). The fate of down-modulated CD4 is also unclear.
Conflicting results have been published indicating either an
increase in the rate of lysosomal sorting and degradation
(Aiken ef al., 1994; Rhee & Marsh, 1994b; Sanffidson et al.,
1994), or an accumulation of CD4 in early endosomes
(Schwartz et al., 1995) or the Golgi apparatus (Brady et al.,
1993).
The apparent specificity and novel mechanism of CD4
down-modulation by Nef have led to the suggestion that a
direct physical interaction between the two proteins might
play a role in the process. Such an interaction has been detected
in insect cells coinfected with recombinant baculoviruses
expressing CD4 and Nef (Harris & Nell, 1994) and more
recently using the yeast two-hybrid system (Rossi et al., 1996).
The sequence requirements for the interaction in insect cells
mirror those necessary for down-modulation in mammalian
cells, i.e. Nef myristoylation and the cytoplasmic domain of
CD4 were critical. However, in the yeast two-hybrid assay Nef
was not myristoylated suggesting that the role of myristoylation may simply be to target Nef to the plasma membrane for
interaction with CD4, rather that playing a more direct role in
the interaction.
This interaction has not been observed to date in a
mammalian context, but in insect cells CD4 is not downmodulated, either by Nef or by phorbol myristate acetate
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(PMA), so clearly a critical component of the mammalian
endocytic machinery is missing. One possible explanation for
this is that glycosylation in insect cells is restricted compared
to mammalian cells,as insect cells lack the enzymes required for
the generation of complex glycosylation patterns (Davies,
1995). Specific complex glycosylation events have been shown
to be involved in targeting, at least in the secretory pathway
(Fiedler & Simons, 1995). For example N-glycans on lysosomal
enzymes are specifically modified to generate a mannose 6phosphate residue that is recognized by a receptor in the transGolgi that sorts proteins into the lysosomal pathway. It might
therefore be the case that in baculovirus-infected insect cells
heterologous glycoproteins would not be endocytosed because of the lack of specific modifications.
The observation of a direct interaction in both the
baculovirus and yeast two-hybrid systems suggests that in a
situation where down-modulation can occur (i.e. mammalian
cells) the interaction may be transient and could serve merely
to recruit CD4 into coated pits from whence endocytosis can
occur. The formation of a Nef-CD4 complex might expose
endocytosis signal(s) either within the cytoplasmic domain of
CD4 (e.g. the dileucine motif) or within Nef. Although serine
phosphorylation of CD4 is not required for Nef-mediated
down-modulation, it is still conceivable that phosphorylation
of Nef might play a role.
The role of Nef-mediated CD4 down-modulation in the
pathogenesis of HIV infection is not clear. The first and most
obvious role would be to prevent lethal superinfection.
Although some in vitro evidence for this has been published for
SIV (Benson et al., 1993) it would seem likely that in order
to efficiently accomplish this Nef would essentially have to
remove all CD4 from the cell surface. Kinetic studies clearly
show that this is not the case, as Nef does not influence the
rates of synthesis or transport of CD4 to the surface, merely
increases the rates of endocytosis and degradation. A more
plausible role, given that HIV infection results in a progressive
qualitative as well as quantitative decline in the CD4 +
population, would be to interfere with the function of CD4 +
cells. There are two ways in which CD4 down-modulation
could result in impaired function. Firstly, by inhibiting
interactions between CD4 + cells and antigen-presenting cells
(APCs). The interaction between the extracellular domains of
CD4 and non-polymorphic regions of MHC class II molecules
on APCs strengthens and stabilizes the interaction between
the T cell receptor (TCR) and class II molecules. By reducing
the surface CD4 concentration, Nef might prevent activation
of T cells through the TCR or result in the transduction of an
inappropriate signal. Secondly, it is believed that CD4 can
transduce signals independently of the TCR. Down-modulation would clearly interfere with these signals and in this
context there are a number of reports that CD4 can transduce
signals that negatively regulate virus replication. Such an effect
has been observed following incubation of infected cells with
anti-CD4 antibodies that bind to the CDR3-1ike region, but not
with anti-CDR2 antibodies (Benkirane et at., 1993). In addition
interleukin-16 (IL-16), one of the soluble factors secreted by
CD8 + cells that has been shown to inhibit virus replication, is
a natural ligand of CD4 and might therefore exert its antiviral
effect through CD4 (Baser et al., 1995).
Recently, Nef has also been shown to induce the endocytosis of MHC class I molecules both in infected U937 cells
and in stably transfected T cells (Schwartz et aI., 1996). The
mechanism of this effect appears to be similar to that involved
in CD4 down-modulation, in that rates of synthesis and
transport through the ER and Golgi apparatus were unaffected
whereas surface MHC class I molecules were rapidly endocytosed, accumulated in endosomes and degraded in lysosomes. There is little sequence homology between the
cytoplasmic domains of CD4 and MHC class I heavy chain,
suggesting that Nef might recognize some common structural
feature. Whatever the underlying mechanism, it is clear that
endocytosis of MHC class I by Nef early in infection would
allow the virus to evade the cytotoxic arm of the immune
response. This is an important new observation that needs to
be confirmed by other laboratories as it will clearly contribute
to the requirement for Nef in pathogenesis in vivo.
(ii) Enhancement of virus infectivity
Although early studies suggested that Nef had a negative
influence on virus replication, it is now clear that, on the
contrary, Nef acts to augment virus replication. The hypothesis
that Nef was a negative factor was refuted in several distinct
stages. Firstly, observations from two laboratories, published
in late 1989, indicated a failure to observe any differences
between the in vitro growth characteristics of isogenic Nef+
and Nef- viruses (Hammes et al., 1989; Kim et al., 1989). This
was followed by two more papers that defined nef alleles
isolated direct from patient material (de Ronde et al., 1992) or
from a virus isolate with a short history of in vitro culture (ELI)
(Zazopoulos & Haseltine, 1993) that could enhance virus
replication in primary lymphocytes and, in the case of ELI Nef,
in T cell lines. In contrast, Nef from the IIIB strain of HIV-1,
which has a long history of in vitro culture, retarded replication
in a T cell line.
The next stage in the development of this aspect of Nef
function was the observation (again in two papers published
together) in early 1994 that although Nef+ and Nef- viruses
replicated equally well in immortalized T cell lines, a requirement for Nef could be demonstrated following low
multiplicity infection of primary quiescent PBMCs that were
subsequently activated by lectin treatment (Spina et al., 1994;
Miller et al., 1994). Under these conditions, Nef+ virus
replicated 13-15-fold more efficiently than Nef- virus, with
peak levels of p24 production between 15-20 days postinfection (p.i.). In contrast, the Neff viruses were producing
barely detectable levels of p24 at 30 days p.i. Nef+ virus was
also more efficient at infecting prestimulated PBMCs, but in
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this case the difference between Nef + and Nef- viruses was not
as marked. Significantly, the differential between Nef+ and
Nef- viruses was not observed following high multiplicity
infection, suggesting that the early experiments that showed a
negative or no effect of Nef were an artifact of the experimental
conditions used, i.e. high multiplicity infection of immortalized
cell lines. The authors argued convincingly that the conditions
of low multiplicity infection of quiescent cells that they
employed more closely resembled the situation in vivo. They
further demonstrated that the effect of Nef was to increase the
infectivity of virions: after normalization for p24 levels, virus
supernatants from cells infected with Nef+ viruses gave 13-17fold more blue foci when assayed in a single-cell infection
assay (HeLa-CD4-LTR-fl-gal cells) than Nef viruses (Miller et
a]., 1995).
Within the last year some insights into the molecular
mechanism of this enhancement have been published. The
observation that Nef- virus could be rescued to wild-type
levels of infectivity by expression of Nef in trans in the cell line
producing the virus, but not in the target cell line, indicated
that Nef in some way influences the production of virus
particles (Miller et al., 1995). An env-defective HIV-1 pseudotyped with amphotropic retrovirus envelope was also responsive to Nef, correlating with earlier data showing that
CD4 was not required for Nef-mediated enhancement and
suggesting that the Nef effect was at the level of core
formation (Miller et al., 1995; Aiken & Trono, 1995).
Myristoylation of Nef was required for enhancement, suggesting that membrane association plays a critical role in the
process (Chowers et al., 1994). Levels of major viral structural
proteins and viral RNA were unaffected by Nef and virus entry
assays indicating that Nef- virus entered the cell normally.
However, recent studies have shown that reverse transcription
of Nef- viruses is impaired (Chowers et al., 1995; Aiken &
Trono, I995). The biochemical details of this role of Nef
remain obscure as it is clear that it does not involve a simple
lack of virion-associated reverse transcriptase activity (as
judged by in vitro assays on virus supernatants) but in some
way optimizes reverse transcriptase activity in infected cells,
presumably in the preintegration complex (Stevenson, 1996).
One could speculate at length as to the possible mechanism
behind this effect of Nef on reverse transcription, but one
obvious possibility is that Nef itself is present in the virion. By
virtue of myristoylation, it is likely to colocalize with Gag and
Gag-Pol at the plasma membrane and could therefore be either
actively or passively incorporated into virions. As yet there are
no data to unambiguously prove or disprove this hypothesis.
Alternatively, Nef might recruit cellular factors such as kinases
to the assembling virus particle. In this regard, a proline-rich
motif in Nef that is capable of interacting in vitro with the SH3
domains of various Src family tyrosine kinases, in particular
Hck and Lck, has been recently identified (Saksela eta]., 1995).
Mutations within the proline-rich motif that abolished the
ability to interact with SH3 domains also abolished en-
_)38L
hancement of the virus infectivity function of Nef when
engineered back into an infectious provirus (Saksela et al.,
1995; Goldsmith et al., 1995). Interestingly, this mutant
retained the ability to down-modulate CD4, suggesting that
these two functions of Nef are distinct. However, a mutant
with the opposite phenotype has yet to be identified. Thus it
is still a formal possibility that CD4 down-modulation is linked
to enhancement of virus infectivity in vivo.
Nef has also been shown to be functional in enhancing both
virus replication and pathogenicity of HIV-1 in a SCID-Hu
mouse system (Jamieson et al., 1994; Aldrovandi & Zack,
1996): Virus loads were 30--40-fold lower in human fetal
thymus/liver implants infected with Nef-deleted viruses as
compared with those infected with wild-type viruses. Additionally, Nef-deleted viruses failed to deplete CD4 + thymocytes in the implants, unlike wild-type viruses. Although the
defect in virus replication could be largely overcome by
increasing the inoculum, this increase did not result in wildtype pathogenicity (Aldrovandi & Zack, 1996), suggesting
that Nef may contribute to the pathogenesis of AIDS
independently of its role in enhancing virus replication by
contributing to the depletion of CD4 + T cells in infected
individuals. Whether the cytotoxic or cytostatic effects of Nef
in tissue culture observed by a number of groups [see section
(iv) below] contribute to this in vivo effect remains to be
determined. Further indirect evidence for the involvement of
Nef in HIV-I pathogenicity in vivo comes from the observation
of Nef deletions in two sets of long-term non-progressor
(LTNP) patients (Kirchoff et at., 1995; Deacon et at., 1995),
suggesting that long-term non-progression may result from
infection with an attenuated (Nef-deleted) HIV-1. However,
another study (Huang et al., I995 a, b) failed to observe either
deletions or functional defects in the Nef genes isolated from
a cohort of LTNP patients. Nef-deletion is thus clearly not the
sole cause of long-term non-progression, although it may
contribute.
In a historical context it is interesting that the about-turn on
the perceived function of HIV-1 Nef from negative to positive
factor coincided with the first reports of a requirement for Nef
in pathogenesis of SIV infection in vivo. In hindsight it did seem
unlikely that HIV would possess a gene product whose
function was to suppress virus replication and clearly as soon
as it was demonstrated that SIV Nef had a fundamental role to
play in vivo much effort was spent demonstrating that the same
situation existed for HIV Nef. As in vivo experiments were out
of the question for HIV (with the exception of the SCID-Hu
mouse system) demonstration of an in vitro correlate was
required. At this point it would seem appropriate to discuss the
SIV Nef story in more detail.
(iii) The critical requirement for SIV Nef in virus
pathogenesis
At the amino acid level SIV and HIV-I Nef share only 40%
homology. In addition, SIV Nef is 50-60 amino acids longer
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than HIV-I Net. Although the genes are at similar positions on
the viral genome, unlike HIV-1 the SIV Net open reading flame
overlaps with the 3' end of env. Therefore, it should not be
taken for granted that the two proteins are functionally
identical. The fact that the sequence and organization of SIV
Net is closer to that of HIV-2 means that these differences are
relevant in a purely human context. A number of early papers
attempted to look at the function of SIV Net in vitro by
performing analogous experiments to those carried out for
HIV Net. Unsurprisingly, as for HIV, these yielded conflicting
results: two reports that deletion of Net from an infectious
provirus generated a virus with enhanced replicative potential
in Cos cells (Niederman et al., 1991) or human Hut78 T-cells
(Binninger el al., 1991) were contradicted by one paper
indicating that deletion of Net had no effect in vitro (Unger et
al., 1992). Although all three papers used SIVmac isolates it
should be noted that the studies demonstrating a negative
effect of Net used SIVmac251 clones, which cause disease,
whereas the third study used clone 1All, which does not
cause disease in macaques. However, all these studies were
superseded by the observation in 1991 that a SIVmac251
proviral clone with a large deletion in Net established an
infection but failed to cause disease in macaques (Kestler et al.,
1991). Virus load in the animals infected with the Net-deleted
virus was 1000-fold lower than wild-type, indicating that Net
was required both for maintenance of high virus load and also
for pathogenesis. Furthermore, when animals were infected
with a provirus that contained a single point mutation in Net
(generating a premature termination codon) they succumbed
to disease with wild-type kinetics. Virus isolation revealed that
the point mutation had reverted, allowing the production of
full-length Net. This observation indicates that viruses expressing a fully functional net gene have a strong growth
advantage in vivo. Other groups have confirmed these findings
(Rud el al., 1992), showing that, in the case of the SIV C8 virus
isolate, in vivo repair of a 12 bp deletion can take place first by
a duplication of an adjacent region and then progressive
mutation of the duplicated sequence such that the translation
product resembles the original amino acid sequence (Whatmore
el al., 1995). These results were a turning point in the history
of Net research, paving the way for a resurgence of interest in
a g e n e product that until then had been considered nonessential. Interest was further fuelled by the observation that
animals infected with the Net-deleted virus were resistant to
subsequent challenge with wild-type virus, suggesting that a
Net-deleted virus might have potential as a candidate attenuated virus vaccine (Daniel et al., 1992; Almond et al., 1995). It
is beyond the scope of this review to consider the pros and
cons of such an approach except to say that, although doubts
have been raised as to the safety of such a vaccine following
reports of the lethality of SIV deletion mutants in neonatal
macaques (Baba et al., 1995), research continues apace.
Recently, Net from pbj14, an acutely pathogenic strain of
SIV that is lethal in pig-tailed macaques within 10 days of
infection, was reported to have transforming ability in NIH3T3
cells as a result of the presence of tyrosine residues at the N
terminus that constituted binding sites for SH2 domains (Du el
al., 1995). Although of interest, it is far from clear whether this
mutant represents an artifact that could not possibly exist in
the wild, especially given the degree of adaptation of SIV to its
host, or an exaggerated manifestation of the true function of
Net. In addition, there is little homology between SIV and
HIV-1 in this region so that this observation cannot explain the
mechanism of action of HIV-1 Net.
(iv) Effects of Nef on cellular signal transduction
pathways
Even before the acquisition of any functional data, a number
of factors (namely the myristoylation of Net and its association
with the plasma membrane and the limited sequence homology
between Net and G-proteins discussed above) prompted
investigators to hypothesize that Net might play a role in
perturbing signal transduction pathways in the infected cell.
The early observations of repression of LTR-driven transcription led to suggestions that Net might be interfering with
the activation of transcription factors involved in LTR function
(Ahmad & Venkatesan, 1988; Yu & Felsted, 1992; Maitra et al.,
1991). This view was supported by a number of reports of
specific inhibition of the inducible transcription factors NF-~cB
(Niederman et al., 1992; Bandres & Ratner, 1994) and AP-1
(Niederman et al., 1993; Bandres &Ratner, 1994) in human T
cells constitutively expressing Net. This effect of Net was
shown to be abolished by mutation of Ala-15 --+ Thr (Bandres
et aI., 1994), in apparent contradiction of an earlier study
(Laurent et al., 1990) which had suggested that Net containing
Ala-15 was inactive as it exhibited a very short half-life, was
not phosphorylated and did not down-modulate CD4. Furthermore, it has been reported that stimulation of T cells
expressing Net with mitogens or anti-TCR antibodies failed to
elicit activation of either transcription factor whereas stimulation by lipopolysaccharide (LPS), tumour necrosis factor
(TNF) or IL-1 was unaffected by Net, suggesting that Net
specifically interfered with a signal emanating from the TCR
(Bandres &Ratner, 1994); however, these results remain to be
corroborated. Both NF-KB and AP-1 are involved in expression
of IL-2 following antigenic stimulation of T cells and two
groups have shown that IL-2 expression is impaired by Net
(Luria el a]., 1991; Collette et al., 1996a). Luria el al. (1991)
demonstrated that mutation of three N-terminal amino acids
(Ala-15 -+ Thr, Gly-29 --* Arg and Val-33 --+ Ala) abolished
this impairment. Again, this observation has been questioned
(Schwartz et aI., 1992) and awaits confirmation.
In an attempt to analyse these effects of Net at the
biochemical level, Net was expressed as a fusion with the
extracellular and transmembrane regions of CDSe in Jurkat T
cells (Baur et al., 1994). Only a small proportion of the chimera
was expressed on the cell surface, but when these cells were
activated by anti-TCR antibodies the CDS-Nef chimera
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inhibited both tyrosine phosphorylation of cellular proteins (in
particular PLCyI, TCR( and an unidentified 36 kDa protein)
and calcium flux. However, when a subpopulation of cells that
expressed high levels of the chimera at the surface was selected
out, these ceils appeared to be constitutively activated. They
expressed the activation markers CD69 and IL-2 receptor (IL2R), had regained the induction of tyrosine phosphorylation
after stimulation, and after prolonged culture most cells died as
a result of apoptosis. Despite the intriguing nature of these
observations there are concerns about the relevance of the
CDS-Nef chimera to the true function of Nef. The cells were
clearly under selection pressure to avoid expression of the
chimera at the surface (as shown by the generation of
premature truncations in Nef after prolonged culture and the
difficulties encountered in initial selection of clones).
Other effects of Nef on signal transduction pathways have
been observed. In NIH3T3 cells Nef expression was reported
to inhibit the increase in cytosolic calcium mediated by inositol
1,4,5-trisphosphate (IP~) when IP3 levels were elevated by
treatment with bombesin or platelet-derived growth factor
(PDGF) (De & Marsh, 1994). The physiological significance of
this observation remains to be clarified. However, in this
context it is interesting to note that a number of investigators
have alluded to the cytotoxic or cytostatic effects of Nef
observed when attempting to generate stable cell lines
constitutively expressing Nef (Murphy el al., 1993; Baur et al.,
1994; RyanGraham & Peden, 1995). This effect of Nef has
recently been confirmed using two different inducible vector
systems (S. J. Cooke, unpublished results). Induction of high
levels of Nef expression in HeLa, Jurkat T cells and Raw264.2
murine macrophages resulted in reductions in growth potential.
6. Clues to the biochemistry of Nef function
from the identification of cellular Nefinteracting factors
In line with the general fervour for protein-protein
interactions amongst the entire biological research community,
there has been an increasing number of reports that have
attempted to define the biochemistry of Nef function by
identifying interactions between Nef and cellular proteins (see
Table 1). The first report of Nef-interacting proteins utilized a
myristoylated Nef--GST fusion protein produced in a baculovirus system to identify a subset of proteins in Iysates from
Jurkat T cells that interacted with Nef in vitro (Harris & Coates,
1993). Interestingly, the majority of these proteins were
membrane associated and failed to bind to non-myristoylated
Nef-GST, suggesting that Nef might interact with a specific
membrane complex. Another set of in vitro Nef-interacting
proteins was observed using E. coli-expressed GST-Nef as
affinity reagent (Greenway el al., 1995). By immunoblotting,
four of these proteins were identified as p56Lck, CD4, MAP
kinase/erk-I and p53. It is not possible to deduce from these
data which proteins are interacting specificalIy with Nef and
which are pulled down by association with other proteins.
More convincing evidence for a direct interaction with CD4
came from observations using recombinant baculoviruses and
the yeast two-hybrid system [discussed in section 5(i) above].
The yeast two-hybrid system has been used to identify an
interaction between Nef and a component of non-clathrincoated vesicles, ]/-COP (Benichou el al., 1994). Nef and ]/-COP
were also shown to interact in vitro and in HIV-l-infected cells.
This interaction has also been demonstrated in vitro using
immobilized myristoylated Nef-GST and a Jurkat T cell
extract (Harris, 1995). The functional significance of this
interaction remains to be analysed but it is conceivable that it
might play a role in the endocytosis of CD4 and MHC class I
described above [section 50)]. Benichou et al. (1994) point out
that//-COP, as well as being a component of the cytosol, has
been found in both the Golgi and endosomes, two compartments that have been identified as sites of CD4 accumulation
in Nef-expressing cells (Brady et al., 1993; Schwartz el al.,
1995). However, the mechanism by which an interaction with
//-COP might contribute to the specific effects of Nef on CD4
and MHC class I surface expression is not obvious at this stage.
A significant number of Nef-binding proteins are protein
kinases, both serine/threonine and tyrosine. The CDS-Nef
chimera system was used to demonstrate that Nef coprecipitated with a serine/threonine kinase that phosphorylated two other Nef-binding proteins of molecular mass 62
and 72 kDa (p62 and p72) (Sawai et al., 1994). The identity of
these proteins is as yet undefined; however, the study has been
extended to show that they interact with amino acid sequences
in the central conserved region and also require membrane
localization of Nef (Sawai et al., 1995). Two studies have
shown that, in addition to this unidentified kinase, Nef
associates with, and is phosphorylated by, novel isotypes of
PKC (Ca 2+- and phospholipid-independent isotypes) (Coates
& Harris, 1995; Bodeus et al., 1995). Although both these
studies were performed in vitro using E. coli-expressed
GST-Nef fusion proteins, the similarity between their results
suggests that this is an important observation with relevance
to Nef function in vivo. It should be stated that conclusive
evidence for serine phosphorylation of Nef in vivo has not yet
been reported, either in Nef-expressing cell lines or in infected
cells. This should not be seen as definitive evidence that Nef is
not phosphorylated since it is possible that Nef phosphoryiation will be transient and may require specific signals to be
delivered to the cell. Resolving this issue is clearly a priority for
the future.
The recognition that Nef possesses a consensus SH3binding proline repeat motif (amino acids 69-78) that is well
conserved amongst Nef isolates led to the observation that this
motif was able to participate in interactions with members of
the Src family of non-receptor tyrosine kinases. Using filterbinding assays and plasmon resonance it was shown that the
Nef proline motif bound to the SH3 domain of Hck with high
affinity (KD 250 nM for intact Nef but 300-fold weaker for the
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Table 1. Nef-interacting proteins
Nef-interactin 9 protein
Cell type/Nef expressionsystem
p280, p97, p75, p57, p55, p32, Jurkat T cells, baculovirus-expressed
p28, p26
myristoy[atedNef-GST
p70, p60, p56, p44, p36, p32, MT-2 and PBMCs, E. coli-expressed
p30, p28, p27 (including
GST-Nef (non-myristoylated)
p56Lck, CD4, MAP kinase
and p53)
Novel protein kinase C
Jurkat and HeLa cells, E. coli-expressed
isotypes
GST-Nef (non-myristoylated)
Uncharacterized
Jurkat T cells expressing CDS-Nef chimeric
serine/threonine kinase(s)
construct
CD4
Coexpression with Nef-GST in insect cells
Yeast two-hybrid system
p56Lck
Baculovirus-expressedLck and Nef-GST.
Jurkat T cell-derivedLck and Nef, isolated
GST-SH2 and SH3 domain and Nef proline
motif
p59Hck
E. coli-expressed GST-SH3 domain fusions
and GST-Nef fusions, U937-derived Hck
p60Src
Coexpression in Cos-7 cells
Serine phosphorylated p62
Jurkat T cells expressing CDS-Nef
and p72
chimeric construct
fl-COP
Jurkat T-cells, yeast two-hybrid system
Jurkat T cells, baculovirus-expressed
myristoylated Nef-GST
isolated PxxP motif) (Saksela et al., 1995 ; Lee et al., 1995). An
additional PxxP motif at amino acids 147-150 was also
implicated in the interaction, possibly explaining the decreased
affinity of the isolated PxxP motif. No interactions with other
SH3 domains, namely Lck and Fyn, were observed. In partial
contradiction to this, a specific interaction with the SH3
domain of Lck, with no binding to Fyn, PI3K and Grb2 SH3
domains has been documented (Collette et al., 1996b).
Although these interactions were easily demonstrated using
isolated protein domains it was also important to show that
they could occur in the context of the native proteins. Both
groups showed that this was the case: Hck could be
precipitated from extracts of U937 cells by immobilized
GST-Nef (but not by a proline motif mutant form) and Nef coimmunoprecipitated with Lck from Jurkat cells expressing Nef.
The interaction between Nef and Lck has been shown to be
dependent on Nef myristoylation (Harris, I995) but does not
require Lck myristoylation (M. Harris, unpublished results).
Binding of Lck to Nef in vitro was inhibited by the presence of
sodium orthovanadate (a tyrosine phosphatase inhibitor) in the
lysis buffer, suggesting that Nef only bound to Lck that was in
the active configuration, i.e. Tyr-505 dephosphorylated. In
addition, Lck bound to Nef failed to autophosphorylate,
suggesting that Nef binding to Lck inhibits kinase activity,
possibly by occluding the kinase site (M. Harris, unpublished
observations).
Reference
Harris & Coates (1993)
Greenway eta]. (1995)
Coates & Harris (1995)
Bodeus et al. (1995)
Sawai et al. (1994)
Harris & Neil (1994)
Rossi et al. (1996)
Harris (I995)
Collette et al. (1996b)
Saksela et al. (1995)
Duet al. (1995)
Sawai et al. (1994)
Benichou et al. (1994)
Harris (1995)
In addition to the interaction between the Nef proline motif
and the Lck SH3 domain it was reported that Nef was tyrosine
phosphorylated and interacted with the Lck SH2 domain
(Collette et al., 1996 b). The site(s) of tyrosine phosphorylation
have not yet been identified but it is intriguing to note that a
tyrosine at position 127 is situated in a region of homology to
the C-terminal regulatory tyrosine (505) of Lck (Nef, YI~7-T-PG-P; Lck, y~05_Q_p_Q_p). The phosphorylated tyrosine in Nef
could potentially compete for binding to the Lck SH2 domain.
A functional interaction between Nef and Lck could help to
explain the role of Nef in AIDS pathogenesis. Lck is critically
important for T cell activation and development (Anderson et
al., 1994 b) and it has been well documented that Lck-deficient
T cell lines are severely compromised in early events such as
calcium mobilization as well as downstream events such as IL2 production. One model for Lck function is that it phosphorylates activation motifs (ITAMs) in the cytoplasmic domain of
the l-chain of an engaged TCR complex which then bind to
another tyrosine kinase, ZAP-70. ZAP-70 is in turn phosphorylated and activated by Lck and proceeds to phosphorylate
downstream effector molecules. By binding active Lck and
inhibiting its kinase activity, Nef would effectively block this
process. The inhibition of Lck function should be considered in
the context of CD4 down-modulation by Nef: CD4 downmodulation results in the activation of Lck and its concomitant
dissociation from CD4, thus allowing it to interact with other
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:38~
cellular proteins including the TCR. One plausible model for
Nef function in T cells is that Nef interacts with and downmodulates CD4, thus preventing the transduction of negative
signals for virus replication [see section 5(i) above]. As a result
of down-modulation Lck is activated and released from CD4.
Nef then interacts with activated Lck and inhibits its kinase
activity, thus preventing downstream phosphorylation events
which would lead to T cell activation.
7. Prospects for antiviral strategies targeted
at Nef
The studies with SIV Nef demonstrate conclusively that
Nef is of critical importance for virus replication and pathogenicity. It follows that inhibition of Nef function would be of
therapeutic benefit and might even allow the development of
antiviral agents that would be efficacious in the long-term,
rather than the short-term benefits that are gained from
presently available anti-retroviral agents such as reverse
transcriptase or protease inhibitors. The reason for suggesting
this is that Nef function requires specific and potentially novel
interactions with cellular components, unlike reverse transcriptase and protease, which act on viral components and are
largely independent of cellular factors. By developing specific
inhibitors of the interactions between Nef and the cytoplasmic
tail of CD4 (Harris & Nell, 1994; Rossi eta]., 1996) or the SH3
domains of the tyrosine kinases p56Lck/p59Hck (Collette et
al., 1996b; Saksela et aI., 1995) the hope would be that Nef
mutations that conferred resistance to these inhibitors would
also be functionally compromised. Clearly, the development of
such inhibitors will require a detailed structure--function
analysis of Nef, enabling these interactions to be precisely
defined in biochemical terms. Although such information is not
yet available progress is being made: in particular, the recent
publication of a solution structure of Nef determined by NMR
(Grzesiek et al., 1996) will be invaluable in defining interacting
domains. For example, this report shows that the two separate
PxxP motifs (amino acids 69-78 and 147-150) predicted to
cooperate in SH3 binding (Saksela et al., 1995) are in fact
closely located in the three-dimensional structure allowing the
delineation of an SH3 domain-binding surface. The novel
nature of the interactions between Nef and cellular proteins
may facilitate the design of inhibitors that do not interfere with
normal cellular functions. Support for such a concept comes
from preliminary data suggesting that the requirements for the
interaction of Nef with the cytoplasmic domain of CD4 appear
to be distinct from those required for interaction of p56Lck
with CD4, insofar as mutations of the cysteines at residues
420/422 abolish Lck binding but have no effect on Nef binding
(M. Harris, unpublished observations).
Nef is a highly immunogenic protein and HIV-infected
individuals frequently exhibit strong antibody (Cheingsong
Popovet al., 1990; Ranki et al., 1990) and CTL responses
(Culmann et al., 1991; Hadida et al., 1992) to Nef. Evidence
!38~
from macaques immunized with vaccinia virus recombinants
expressing Nef suggests that Nef-specific CTLs are protective
(Gallimore et al., 1995), as an inverse correlation between the
Nef-specific CTL precursor frequency and virus load was
observed. Thus it seems likely that these responses could be
involved in the control of infection during the clinical latency
phase. Enhancing the immune response to Nef might therefore
be a potential therapeutic option that should be considered,
with the proviso that the primary amino acid sequence of Nef
is highly variable making immune escape likely. This type of
approach would therefore need to be targeted to conserved
functional domains of Nef, again invoking the requirement for
a detailed structure-function analysis.
8. Conclusions
Much progress has been made since the identification of
Nef (or 3'off as it was then known) as a protein product of
HIV-1 in 1985 (Allan ef al., 1985). As I have discussed in this
review, a decade of studies has shown that Nef has a number
of clearly defined in vitro biological functions and a critical role
in virus pathogenesis in vivo. There are several tasks to be
undertaken in the foreseeable future. Firstly, the biochemical
mechanisms of Nef function in vitro must be accurately defined,
hopefully leading to the development of chemotherapeutic
antiviral agents. Implicit in this analysis must be the investigation of the influence of Nef on virus replication in cell types
other than T cells that are known to be infected by HIV, e.g.
macrophages and glial cells. It is conceivable that the
biochemistry of Nef function in these cell types may be distinct
from that in T cells. With regard to macrophages it is pertinent
that Nef apparently interacts with a macrophage-specific
tyrosine kinase, Hck, with high affinity (Saksela et al., 1995).
Secondly, the relative contribution of each of the in vitro
functions to the role of Nef in vivo must be identified.
Intuitively, it is to be expected that both CD4 downmodulation and the enhancement of virus infectivity will be
important for pathogenesis of HIV infection. However, formal
proof of this must be obtained. It will also be important to
ascertain whether these in vitro functions are truly independent.
Although both CD4 down-modulation and enhancement of
virus infectivity can occur independently under in vitro
conditions, it cannot be ruled out that they are linked in some
way in vivo. They may be manifestations of an underlying
biochemical mechanism, as yet unidentified. The most obvious
grey area concerning Nef function is the effect of Nef on signal
transduction pathways. This aspect of Nef embraces the most
controversy and disagreement and clear interpretation of the
available data is impossible. Above all, the priority here must
be to demonstrate that any effects of Nef observed in vitro are
relevant to virus replication and not merely artifacts of
expression of a heterologous protein. That is the real challenge
for the future.
I thank Jim Neil, Derek Mann and Mark Roberts for critical reading of
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this manuscript. Although this review was started with the intention of
being an exhaustive survey of the Nef literature, time and space have not
permitted this. I therefore apologize in advance to all those who feel that
their work has been under-represented. Work in the author's laboratory
is funded by the British Medical Research Council.
References
Aderem, A. (1992). The MARCKS brothers: a family of protein kinase
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