JOURNAL OF VIROLOGY, Dec. 2004, p. 13335–13344
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 23
Impact of Nef-Mediated Downregulation of Major Histocompatibility
Complex Class I on Immune Response to Simian
Tomek Swigut,1†‡ Louis Alexander,2† Jennifer Morgan,3Jeff Lifson,4Keith G. Mansfield,3
Sabine Lang,3R. Paul Johnson,3Jacek Skowronski,1and Ronald Desrosiers3*
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1; Department of Epidemiology and Public Health,
Yale Medical School, New Haven, Connecticut2; New England Primate Research Center, Southborough,
Massachusetts3; and Laboratory of Retroviral Pathogenesis, AIDS Vaccine Program, SAIC Frederick
Cancer Research and Development Center, Frederick, Maryland4
Received 26 May 2004/Accepted 2 July 2004
Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV)
and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex
(MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activa-
tion. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that
selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant
forms of SIV exhibited higher levels of CD8?T-cell responses 4 to 16 weeks postinfection than seen in monkeys
infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to
32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate
that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to
downregulate MHC class I contributes importantly to the totality of nef function.
nef is one of six auxiliary genes found in simian immunode-
ficiency virus (SIV) and human immunodeficiency virus (HIV).
Unlike the gag, pol, and env genes, nef is not essential for the
ability of virus to replicate. However, nef does contribute to the
levels of persisting viral replication observed in infected mon-
keys and humans and to the propensity to cause disease. In
contrast to infection by parental nef?SIV, rhesus monkeys
infected with nef-deleted forms of SIV typically maintain low
or unmeasureable levels of postacute viremia, typically ?200
copies of viral RNA per ml of plasma (4, 30). A minority of
monkeys develop moderate postacute viral loads, and some
progress to disease even in the absence of a nef gene (4, 8, 16).
Studies in humans have been facilitated by the discovery of
rare cases of infection by nef-deleted forms of HIV-1 (15, 31).
Most of these humans have exhibited viral loads at or below
the limits of detection for prolonged periods. However, some
have developed moderate viral loads and some have exhibited
declining CD4?lymphocyte counts (22, 35). Thus, the relative
importance of nef and the phenotypic effects of infection with
nef-deleted virus appear to be similar in the SIV/rhesus mon-
key and HIV-1/human systems.
What is the function of Nef that is responsible for these
phenotypic effects? The answer has unfortunately been
clouded by the demonstration of a diverse array of activities
associated with expression of this protein in vitro. These in-
clude downregulation of the CD4 receptor for virus entry (1,
18), downregulation of major histocompatibility complex
(MHC) class I molecules from the surface of cells (47), down-
regulation of other surface transmembrane proteins (10, 55),
and lymphocyte activation to facilitate viral replication (3, 9,
17, 49, 51). The Nef proteins of both SIV and HIV exhibit
these properties. Downregulation of CD4 could minimize
CD4’s ability to interfere with the appearance or function of
envelope on virions (34, 43); it could also help to prevent toxic
effects associated with superinfection.
Downregulation of MHC class I molecules can make in-
fected cells less susceptible to lysis by cytotoxic T lymphocytes,
and it has been proposed that Nef-mediated downregulation of
MHC class I is an important immune evasion mechanism em-
ployed by HIV and SIV (13, 62). However, because downregu-
lation of MHC class I in vitro typically requires relatively high
levels of Nef expression, some have questioned the physiologic
relevance of the phenomenon (28, 62).
The ability of Nef to interact with the zeta chain of the T-cell
receptor (10, 24) is responsible in whole or in part for Nef-
mediated downregulation of CD3 (53). Concerted downregu-
lation of CD3, CD4, and CD28 (55) as well as the ability of Nef
to interact with cellular kinases (44, 45, 50) is probably related
to alterations in a number of lymphocyte signaling pathways,
T-cell receptor signaling in particular (3, 20, 46, 48, 55, 56, 58,
60). The effects of Nef on CD28, CD4, CD3, and MHC class I
expression are all genetically separable by mutations (55).
Because replication of both SIV and HIV occurs maximally
in lymphocytes that are activated, because most lymphocytes in
the body are resting or minimally activated, and because Nef is
an early gene product, early expression of Nef in a minimally
activated cell in vivo may help to maximize the amounts of
* Corresponding author. Mailing address: New England Primate
Research Center, One Pine Hill Drive, Box 9102, Southborough, MA
01772-9102. Phone: (508) 624-8042. Fax: (508) 624-8190. E-mail:
† Co-first authors contributed to similar extents.
‡ Present address: Laboratory of Vertebrate Embryology, The
Rockefeller University, New York, NY 10021.
virus production by increasing the state of cellular activation.
The recent discovery that SIV?nef replication in lymphoid
tissues is restricted to regions of highly activated T lymphocytes
(52) is consistent with this logic. The interleukin-2-dependent
221 cell line has been used to reflect these lymphoid cell-
activating properties of Nef because viral replication in these
cells is heavily dependent upon Nef in the absence of interleu-
Nef has also been reported to induce Fas ligand, and this
could serve to limit immune-mediated killing of infected cells
by the induction of apoptosis in reactive cells responding to the
sites of infection (6, 60, 61). Other activities have also been
described for Nef (39, 56, 57, 59).
Here, we show that Nef does serve to limit the CD8?cellular
response to SIV and that the ability to downregulate MHC
contributes importantly to the totality of Nef function.
MATERIALS AND METHODS
Site-specific mutagenesis. Mutations were engineered with a splice overlap
extension technique (23). PCR fragments containing the desired mutations were
inserted into the pSIV-3? vector (containing SIV nef sequences) with SacI and
EcoRI restriction enzymes (New England Biolabs, Beverly, Mass.). Thorough
DNA sequences of the resultant recombinant vectors verified the proper se-
quences for all mutants; recombinant clones selected for study contained the
exactly desired sequence.
Virus stocks and monkey infection. Stocks of SIV were prepared by transfec-
tion of CEMX174 cells with cloned DNA and harvest of the cell-free supernatant
at or near the peak of virus production 6 to 10 days after transfection as
previously described (27). Monkeys were infected by intravenous inoculation of
SIV diluted in RPMI medium without serum to contain 50 ng of p27 per dose.
The concentrations of p27 were determined by antigen capture with the Coulter
kit according to the manufacturer’s recommendations. Animals were screened
for the presence of the Mamu-A*01 allele with a PCR single-site protocol as
previously described (32).
Rhesus macaques (Macaca mulatta) were housed at the New England Primate
Research Center in a centralized animal biolevel 3 containment facility in ac-
cordance with the standards of the Association for Assessment and Accreditation
of Laboratory Animal Care and Harvard Medical School’s Animal Care and Use
Committee. Animals were tested and found free of simian retrovirus type D,
simian T-lymphotropic virus 1, and herpes B virus before assignment to the
experimental protocol. All procedures were conducted under ketamine anesthe-
sia, and euthanasia of animals was performed when deemed necessary by the
Viral loads. Plasma viral load was determined with a real time reverse tran-
scription-PCR procedure, essentially as described (36). As used in the current
studies, the assay has a threshold of 100 copy equivalents/ml, with an interassay
coefficient of variation of ?25%.
Immunofluorescent staining and flow cytometry. Monomers of biotinylated
Mamu-A*01 class I MHC molecules complexed with the SIV Gag peptide
CTPYDINQM (Gag181-189) were generated as previously described (5). Tetram-
ers were prepared from purified monomers by the gradual addition over 24 h of
allophycocyanin-streptavidin (Molecular Probes) to a 4:1 final molar ratio.
Whole blood or peripheral blood mononuclear cells isolated by Ficoll-sodium
diatrizoate centrifugation (Ficoll 1077; Sigma) were washed with phosphate-
buffered saline containing 2% fetal bovine serum and incubated with conjugated
antibodies and the Mamu-A*01/Gag181-189tetramer for 30 min at 22°C. After
staining, the cells were washed and resuspended in phosphate-buffered sa-
line–2% fetal bovine serum. Antibodies used included fluorescein isothiocya-
nate-conjugated anti-human CD3 (SP34, Pharmingen, San Diego, Calif.), and
peridinin chlorophyll protein-conjugated anti-human CD8 (BD Biosciences,
Mountain View, Calif.). Simultest reagents were used as isotype controls (BD
Biosciences). Analysis was performed with a FACSCalibur flow cytometer (Bec-
ton Dickinson). In general, a total of 200,000 events were acquired, and analysis
of tetramer-staining cells was carried out on CD3?CD8?gated lymphocytes.
Levels of Mamu-A*01/Gag181-189staining in controls consisting of either Mamu-
A*01-negative, SIV-infected animals or Mamu-A*01-positive, SIV-uninfected
animals consistently yielded levels of background staining of 0.05% or less.
Isolation of genomic (cellular) DNA. CEMx174 cells (5 ? 106) infected with
SIV recovered at weeks 16 and 32 postinfection were lysed, and the DNA was
isolated according to the recommendations of the Qiagen QIAamp DNA blood
minikit (Qiagen, Inc., Valencia, Calif.).
PCR amplification of nef sequences. A total of 20 ?l of each DNA preparation
served as the template for PCR amplification. PCR primers flanking the nef locus
of SIVmac239 were chosen for a first round of amplification: F13 (9161 to 9174),
5?-TATATTCATTTCCTGATCCGCCAAC-3?, and R16 (10151 to 10171), 5?-C
CCCAGTACCTCCCCGTAACA-3?. The 100-?l volume reactions contained 10
?l of TaqPlus Long 10x high-salt buffer (Stratagene, La Jolla, Calif.), 5 ?l of 4
mM deoxynucleoside triphosphate mix (Stratagene), 2 ?l of each primer (100
pmol/?l), 1 ?l of TaqPlus Long polymerase (5 U/?l) (Stratagene), and 60 ?l of
distilled sterile water. The PCR samples were heated at 95°C for 5 min, followed
by 30 cycles of PCR amplification in a thermal cycler (MJ Research, Watertown,
Mass.). Each cycle consisted of a 94°C denaturation step for 1 min, a 58°C
annealing step for 1 min, and a 72°C polymerization and elongation step for 3
min, followed by a final elongation step at 72°C for 10 min.
A second round of PCR was employed in order to introduce Xbal and MluI
restriction sites to facilitate cloning into the pCG7CG expression vector. The
primers used to introduce the restriction sites were mac239Nef Xbal-5? (5?-GC
TCTAGACTAAAGATGGGTGGAGCTATTT CCATGA-3?) and mac239Nef
MluI-3? (5?-ACGCGTCAGCGAGTTTCCTTCTTGTCA-3?). The conditions for
the PCR were the same except that 1 ?l of the first-round PCR product was used
as a template. Additionally, the PCR protocol was adjusted to 68°C for 1 min for
the annealing step.
Cloning. PCR products were gel purified with the Qiagen QIAquick gel ex-
traction kit (Qiagen, Inc.) and then cloned with a TA cloning kit (Invitrogen,
Carlsbad, Calif.). Ligated products were transformed into Escherichia coli XL-2
Blue ultracompetent cells (Stratagene) and plated onto Luria-Bertani agar plates
containing ampicillin (100 ?g/ml), 5-bromo-4-chloro-3-indolyl-?-D-galactopyr-
anoside (X-Gal) (40 ?l of a 40-mg/ml stock solution), and isopropylthiogalacto-
pyranoside (IPTG) (40 ?l of a 100 mM stock solution).
Sequencing of nef clones. White colonies were chosen and miniprep DNA was
prepared with the Promega Wizard Plus Miniprep DNA purification system
(Promega, Madison, Wis.). Individual clones containing nef inserts were se-
quenced across the entire insert on a Beckman Coulter CEQ 8000 genetic
analysis system with the dye-labeled dideoxy terminator cycle sequencing kit as
specified by the manufacturer (Beckman Coulter, Fullerton, Calif.). In addition
to individual clone sequencing, the PCR product from each animal was se-
quenced with the recommendations of the dye-labeled dideoxy terminator cycle
sequencing kit for sequencing PCR products (Beckman Coulter). All sequences
were analyzed with Sequencher 4.1 software (GeneCodes Corp., Ann Arbor,
Transient assays of receptor downregulation by Nef. Open reading frames
encoding mutant and variant Nef proteins were subcloned into the pCGCG
bicistronic expression vector containing green fluorescent protein (GFP) under
the translational control of the internal ribosome entry site (37). Jurkat T cells
expressing high levels of human CD4 were maintained and transfected by elec-
troporation as described previously (21, 37). After overnight culture, aliquots of
2 ? 105cells were reacted with saturating amounts of phycoerythrin-conjugated
monoclonal antibody HIT3A (Becton Dickinson) specific for CD3-ε subunit,
phycoerythrin-conjugated monoclonal antibody Leu-3A (Becton Dickinson) spe-
cific for CD4, phycoerythrin-conjugated monoclonal antibody W6/32 (Immuno-
tech) specific for class I MHC complex, or phycoerythrin-conjugated monoclonal
antibody CD28.2 (Pharmingen), specific for human CD28. Flow cytometry anal-
yses for GFP and surface expression of CD4, CD3, CD28, and class I MHC were
performed on Epics Elite or FACSCalibur flow cytometer as described previ-
ously (37, 54).
Properties of MHC-selective mutants. The sequences in SIV
Nef that are required for MHC downregulation have been
mapped to the C-terminal region (54). For the purpose of
experimental monkey infection, we set out to make mutations
that would be difficult to revert and that selectively affected
MHC downregulation. Mutations that resulted in two-amino-
acid deletions within SIV239 Nef residues 238 to 242 selec-
tively affected the ability to downregulate MHC class I (Fig. 1
and 2). The mutant with deletion of Nef residues 239 and 240
13336 SWIGUT ET AL.J. VIROL.
(?239-240) was selected for testing in rhesus monkeys.
Nef?239-240 behaved like the wild-type parental Nef in the
downregulation of CD4, CD3, and CD28 but was negative for
any detectable downregulation of MHC class I (Fig. 2). SIV239
Nef?239-240 also behaved like the parental SIV239 in its abil-
ity to replicate in 221 cells in the absence of interleukin-2 (Fig.
3). The mutant form of SIV239 Nef with a stop signal at codon
238 followed by two frameshift (fs) mutations (Fig. 1) behaved
like Nef?239-240 in the in vitro assays (Fig. 2 and 3). We
therefore forwarded one deletion mutant (SIV239 Nef?239-
240) and the truncation mutant (SIV239 Nef238stop/fs/fs) for
testing in rhesus monkeys.
Phenotype of rhesus monkey infection. Two rhesus monkeys
were inoculated with normalized doses of SIV239 Nef?239-
240 and two were inoculated with SIV239 Nef238stop/fs/fs. As
controls, two monkeys were similarly inoculated at the same
time with the parental SIV239 wild type and two were inocu-
lated with SIV239?nef. SIV239?nef has a 181-bp deletion in
nef beginning at nucleotide 175 of the nef coding sequence that
abrogates all functional activities of Nef. To facilitate quanti-
tation of CD8?T-cell responses with MHC tetramers, all eight
of these monkeys expressed the Mamu A*01 MHC class I
The levels of virion-associated SIV RNA in plasma were
monitored in blood samples taken at various times after the
inoculations (Fig. 4). In the monkeys that received wild-type
SIV239, plasma RNA levels peaked at day 14 (5,600,000 per ml
in animal 414-98) and at day 8 (24,000,000 per ml in animal
189-01). The average of 14.8 ? 106RNA copies per ml at peak
in these two animals is quite close to the average seen previ-
ously in 10 animals (40 ? 106RNA copies per ml) (29). One of
the control animals infected with wild-type SIV239 (189-01)
exhibited postacute viral loads (?3 ? 106copies per ml) at 12
to 30 weeks postinfection that were very similar to what we
typically observed previously (29). However, the other monkey
FIG. 1. Difficult-to-revert mutations in nef that selectively elimi-
nate ability to downregulate MHC class I molecules. WT 239 refers to
the nef sequence present in the wild-type parental SIV239 between Nef
amino acid residues 235 and 244. ?239-240 and ?241-242 are Nef
variants deleted at residues 239 to 240 and 241 to 242, respectively.
238stop/fs/fs is a Nef variant with a stop codon introduced at residue
238 followed by two downstream frameshift mutations.
FIG. 2. Selective disruption of class I MHC downregulation by difficult-to-revert mutations in SIVmac239 Nef. Jurkat T cells were transiently
transfected with pCGCG bicistronic vectors expressing the green fluorescent protein (GFP) marker alone or coexpressing green fluorescent protein
and wild-type (wt 239) or mutant Nef proteins (238stop/fs/fs, ?239-240, and ?239-241). CD28, CD4, CD3-ε, and CD28 class I MHC cell surface
expression and green fluorescent protein fluorescence were quantitated simultaneously by flow cytometry.
VOL. 78, 2004 Nef AND MHC DOWNREGULATION 13337
that received wild-type SIV239 (414-98) was unusual in that it
exhibited postacute viral loads in the range of 300 to 3,000
copies per ml (Fig. 4). The two monkeys that received
SIV239?nef were similar to other such animals that we have
studied previously in that viral loads at peak height (day 8)
were on average 50-fold lower than those seen in animals
inoculated with wild-type SIV239. Viral loads at set point in
the two monkeys infected with SIV239?nef were also typical in
that they were near or below the limit of detection (Fig. 4).
Viral loads at peak height in the four animals that received
the MHC-selective mutants were indistinguishable from those
seen in the wild-type SIV239-infected animals (Fig. 4). They all
peaked at day 8 with an average of 52 ? 106copies/ml (range,
12? 106to 70 ? 106copies per ml). Thus, there appeared to be
no inherent defect in the ability of these mutated strains to
replicate in monkeys. The four monkeys that received the
MHC-selective nef mutants appeared to exhibit lower viral
loads 4 to 14 weeks postinfection than the viral loads seen in
SIV239-infected control monkey 189-01 over this time range.
However, the differences were not statistically significant when
compared to the two Mamu A*01-positive control monkeys
used in this study. When viral loads from four additional wild-
type-infected Mamu A*01?monkeys from previous studies
were included in the analysis, statistically significant differences
in viral loads were again not observed at any time point com-
pared to the viral loads in the four monkeys that received the
MHC-selective nef mutants.
Immune responses in infected rhesus monkeys. The ability
of CD8?T lymphocytes from infected monkeys to react with
antigenic peptide and MHC I tetramer corresponding to the
Days post infection
FIG. 3. SIV239 with MHC-selective mutations replicates like wild-
type parental SIV239 in 221 cells in the absence of interleukin-2. SIV
containing 10 ng of p27 was used to infect 221 cells in the presence of
5% serum without interleukin-2 as previously described (4). Virus
production in the cell-free supernatant was monitored on the indicated
days by assay of levels of p27 antigen. Error bars are based on qua-
FIG. 4. Viral loads in monkeys infected with wild-type (WT) SIV239 or SIV239 mutated in Nef. Dashed lines indicate the threshold sensitivity
of the assay (100 copies/ml). Mm, M. mulatta.
13338 SWIGUT ET AL.J. VIROL.
Mamu-A*01-restricted Gag181-189epitope CTPYDINQM was
monitored. The four monkeys infected with the SIV strains
carrying the MHC-selective nef mutations exhibited higher lev-
els of CD8?cellular responses than the two monkeys infected
with the parental wild-type SIV239 at 4 to 14 weeks after
infection (Fig. 5A and B) (P ? 0.06 by Mann-Whitney U test at
weeks 4 and 9). Differences became less discernible by 16 to 20
weeks postinfection. To increase the power of the analyses, we
separately included tetramer data from two other Mamu-
A*01?, SIV239-infected monkeys from another study for
which such data were available. Again, all four monkeys in-
fected with the SIV strains with MHC-selective nef mutations
made Gag tetramer responses 4 to 14 weeks after infection that
were consistently higher than those seen in the four wild-type
SIV239-infected monkeys. These differences were statistically
significant (P ? 0.02, 0.04, and 0.02 at weeks 4, 8 to 9, and 13
to 14 after infection, respectively, by Mann-Whitney U test).
Sequence evolution. We next charted the evolution of se-
quence changes in nef over time. nef sequences present at 16
and 32 weeks postinfection were amplified by PCR of DNA
from cells infected with recovered virus. Direct sequencing of
the amplified products gave a sense of the predominant amino
acid present at each location, and sequencing of individual
clones gave a sense of the range of variation within each pop-
ulation at those times. Little variation was observed in the
week 16 samples from the two control animals infected with
wild-type virus; two positions of amino acid variation were
observed in the bulk population in monkey 414-98 at that time,
and none were observed in monkey 189-01.
In contrast, greater levels of variation were observed in the
monkeys infected with the MHC-selective mutants at 16 weeks.
All four of these monkeys retained their original Nef?239-240
or Nef238stop/fs/fs mutation, which is not surprising given the
expected difficulty in generating revertants of these mutations.
However, these four monkeys exhibited three to six predicted
amino acid variations per nef gene in the sequence of the bulk
population and three to eleven predicted amino acid variations
in individual clones. Sequence changes noted in the bulk pop-
ulation usually appeared in the individual clones. Examples of
predicted amino acid sequence changes in individual clones
from the four animals are shown in Fig. 6. The two monkeys
infected with SIV239 Nef?239-240 (140-98 and 20-01) showed
a clustering of amino acid changes within the region spanning
residues 11 to 36, and the two monkeys infected with SIV239
Nef238stop/fs/fs (146-98 and 183-01) showed a clustering of
amino acid changes within the region spanning residues 101 to
112 at week 16 (Fig. 6).
One of the striking features of the nef sequence variation in
the four animals infected with the MHC-selective mutants was
that many of the variations changed the amino acid to that
observed in the consensus sequence for the Nef protein of
HIV-1. More than 50% of the amino acid changes in Nef
observed at 16 weeks changed the amino acid to the HIV-1 Nef
consensus at this residue. These included E36K, S101P, S112T,
S161P, E190K, and Y229H. The S101P change created an
additional PXXP motif, as is present in most HIV-1 Nef se-
quences and absent in all SIVmac and SIVsm sequences (33).
By 32 weeks, the nef sequences had evolved even further in
the monkeys infected with the MHC-selective mutants. One
monkey, 140-98, had to be sacrificed because of deteriorating
health prior to 32 weeks, but nef sequences were obtained from
recovered SIV from the other three animals at 32 weeks.
Again, all clones retained the original Nef?239-240 or
Nef238stop/fs/fs mutation from the appropriate animals. Week
32 clones exhibited 7 to 12 amino acid changes per nef gene in
various clones. Certain signature changes present at both
weeks 16 and 32 in the same animal confirmed the validity of
the sequence determinations. For example, S161P was ob-
served in the majority of clones at weeks 16 and 32 in monkey
146-98 but was not observed in other animals. E36K was ob-
served with reasonable frequency in both week 16 and week 32
clones from monkey 20-01, but this change was not observed in
other animals. S112T was observed in the majority of clones at
weeks 16 and 32 in monkey 183-01, was observed in animal
146-98 only at week 32, and was not observed in the other
animals. K105R and Y229H predominated in monkeys 146-98
and 183-01 at both 16 and 32 weeks but did not appear in
monkey 20-01 until 32 weeks.
Thus, a pattern of sequence changes was observed that
showed some commonality among all animals infected with the
MHC-selective nef mutants but even more striking common-
ality based on what the individual animal had received for the
SIV inoculum. Thus, common changes such as S101P and
Y229H occurred in all four animals infected with the MHC-
selective mutants, and other signature changes arose specifi-
cally with Nef?239-240 or with Nef238stop/fs/fs. Amino acid
substitutions in Nef in these four animals accumulated at an
FIG. 5. Viral Gag-specific CD8?cellular responses measured by
Gag tetramer staining. (A) Results for individual animals. (B) Aver-
ages from the four monkeys infected with MHC-selective SIV mutants
versus averages from the two control monkeys infected with parental
SIV239. Numbers refer to the percentage of CD3?CD8?Gag181-189
VOL. 78, 2004Nef AND MHC DOWNREGULATION 13339
astonishing rate of about 7% per year, an extremely unusual
high rate (12, 26).
Sequence changes restore ability to downregulate MHC
class I. Full-length nef clones obtained at 16 and 32 weeks were
inserted into the expression vector and used in downregulation
assays. Of 11 nef clones obtained at week 16 from the four
monkeys, 8 showed various levels of MHC-downregulating ac-
tivity (Fig. 7). None of these eight showed MHC-downregulat-
ing activity as strong as that of the parental SIV239 Nef. The
five nef clones obtained at week 16 from the two monkeys
infected with SIV239 Nef238stop/fs/fs (monkeys 146-98 and
183-01) in particular showed a consistent, intermediate gain-
of-function phenotype. Of 16 nef clones analyzed from week
32, 15 showed readily detectable MHC-downregulating activity
(Fig. 7). There was a clear progression from week 16 to week
32 in the ability to restore MHC-downregulating activity. Eight
of the 16 nef clones from week 32 showed MHC-downregulat-
ing activity that was indistinguishable from or only slightly less
than that observed with the parental SIV239 Nef. Four of the
ten nef clones obtained at week 16 showed loss of CD28 down-
regulating activity, but by week 32 14 of 16 clones analyzed
exhibited CD28-downregulating activity that was comparable
to that of the parental SIV239 Nef. The vast majority of clones
at both 16 and 32 weeks exhibited CD4- and CD3-downregu-
lating activity that was indistinguishable from or only slightly
less than that observed with the parental SIV239 Nef.
A surprising number of functional activities have been ob-
served for Nef. Lymphoid cell activation and downregulation
of CD4, CD3, CD28, and MHC class I have been assayed in
our current experiments. In addition, infectivity enhancement
and induction of Fas ligand have also been described as func-
tional activities (40, 61). Nef has also been reported to induce
lymphocyte chemotaxis toward infected macrophages (57) and
to inhibit proapoptotic signaling in infected cells (19). Infec-
tivity enhancement may relate in whole or in part to CD4
downregulation (34, 43) and increased cellular activation (3,
20, 46). Induction of Fas ligand has been suggested to increase
apoptotic death of reactive cells trying to respond to infection
The goal of our current studies was to obtain evidence for
the contribution of one activity of this multitude, MHC class I
downregulation, to the ability of SIV to replicate in rhesus
monkeys. Although the MHC-selective Nef?239-240 and
Nef238stop/fs/fs mutations had no effect on lymphoid cell ac-
tivation in the 221 cell assay or on downregulation of CD4,
FIG. 6. Sequence changes in Nef 16 and 32 weeks after infection. Circles identify the locations of amino acids that correspond to the HIV-1
consensus amino acid.
13340 SWIGUT ET AL.J. VIROL.
CD3, or CD28, we do not know whether these mutations
affected other known or unknown activities. Nonetheless, the
stronger CD8 responses in mutant-infected animals and the
strong selection for unusual compensatory sequence changes
that restored MHC-downregulating activity provide convincing
evidence for a contribution of this activity to the persistent
replication of SIV in rhesus monkeys. Although a number of
other viruses encode gene products that interfere with MHC
expression (7, 11, 14, 25, 42, 63), we are aware of only one
other analogous report in which a functional role in immune
evasion has been validated in an animal system as we have
done here. Stevenson et al. (51a) knocked out the class I
FIG. 7. Variant Nef sequences at 16 and 32 weeks postinfection exhibit restoration or partial restoration of the ability to downregulate MHC
class I. Analysis of sequences recovered from monkeys that received SIVmac239 carrying the Nef238stop/fs/fs (A) or the Nef?239-240 (B) mutant.
Analysis was performed as described in the legend to Fig. 2.
VOL. 78, 2004 Nef AND MHC DOWNREGULATION13341
MHC-downregulating K3 gene from murine gammaherpesvi-
rus 68 and found stronger virus-specific CD8?T-cell responses
and lower viral loads in mice.
Mutations became fixed in the population of Nef sequences
in monkeys infected with the MHC-selective mutants at an
astonishing rate, resulting in about 7% amino acid changes per
year. Even the highly variable gp120 envelope protein accu-
mulates sequence changes at a rate of only about 2% per year
(12, 26), more than three times less than the rate seen in Nef
in the monkeys infected with the MHC-selective mutants in the
current study. This suggests strong selective pressure for the
appearance of sequence variants and strong selective advan-
tage of the mutant forms that became fixed in the population.
In a previous study (41), a simple point mutation that elimi-
nated MHC-downregulating activity reverted within a few
weeks of monkey infection. In all clones analyzed at 16 and 32
weeks after infection in our current study, the difficult-to-
revert Nef?239-240 and Nef 238stop/fs/fs mutations were re-
tained, forcing the virus to try to compensate by changing
residues elsewhere in the Nef protein.
The changes in Nef progressively increased the ability to
downregulate MHC from weeks 0 to 16 to 32 such that by week
32 the ability to downregulate MHC class I was similar to that
of the parental SIV239 Nef. Based on the large number of
sequence changes and the time required to restore full MHC-
downregulating activity, the virus was clearly under strong se-
lective pressure over a prolonged period to restore the activity;
this provides unambiguous evidence for the contribution of
MHC downregulation to the ability of SIV to replicate in
monkeys. The ability to restore the activity by sequence
changes quite distant in the linear sequence is testimony to the
remarkable flexibility and multiplicity built into the Nef protein
Surprisingly, despite similar functional activities and about
30% sequence identity at the amino acid level, HIV-1 nef
differs from SIVmac nef in the location of mutations that will
selectively knock out the ability to downregulate MHC class I
(54). Several groups have shown that mutation of HIV-1 nef in
the vicinity of the coding sequences for PXXPXXPXXP, cor-
responding to residues 101 to 110 in Fig. 6, will selectively
knock out the ability to downregulate MHC class I (21, 38, 54).
In contrast, Swigut et al. (56) and our data here show that
MHC-selective mutations map to the C-terminal region of
SIV239 Nef. Minimal amino acid changes that restored MHC-
downregulating activity to Nef?239-240 and Nef238stop/fs/fs
are interestingly located in the same general vicinity in the Nef
linear sequence as the MHC-selective mutations in HIV-1 Nef.
In fact, the S101P change seen in SIV Nef in all four animals
in our current study creates a PXXPXXP sequence that in
HIV-1 is essential for HIV-1 Nef’s ability to downregulate
MHC class I. Amazingly, more than 50% of the amino acid
changes in SIV Nef in the monkeys infected with SIV239
Nef?239-240 and SIV239 Nef238stop/fs/fs resulted in the con-
sensus HIV-1 amino acid at that location. This could be viewed
as an accelerated, partial recapitulation of the evolution that
occurred naturally over millennia in the emergence of HIV-1.
A converse evolution of HIV-1 to SIV-like nef sequences was
observed previously in monkeys infected with a recombinant
SIV containing an HIV-1 nef gene (SHIVnef) (2).
CD8?T-cell responses were significantly higher in monkeys
that received the MHC-selective mutant SIVs than in monkeys
that received parental SIV239, from 4 to 14 weeks postinfec-
tion. It seems likely that the evolution of sequence variants
with restored or partially restored MHC-downregulating activ-
ity was responsible for decreased ability to discern effects on
the number of virus-specific CD8 cells at later time points.
Stronger CD8?antiviral responses would be expected to trans-
late into lower viral loads, but we were unable to demonstrate
any statistically significant differences in viral loads among the
groups, excluding of course the SIV?nef animals. It seems
likely that the evolution of restored MHC-downregulating ac-
tivity minimized the magnitude and duration of any effects of
the mutations on viral load. Other factors may also have con-
tributed. The number of monkeys employed was necessarily
limited, and significant differences may have been observed
with much larger numbers of monkeys.
A controlling immune response is likely to consist of multi-
factorial components; the effects of the MHC activity of Nef
influenced the levels of only one of these multifactorial com-
ponents, virus-specific CD8?cellular responses. SIV destruc-
tion of virus-specific CD4?helper cells may have limited the
effectiveness of increased numbers of virus-specific CD8?cells
that resulted from the loss of MHC-downregulating activity.
Nonetheless, although we were not able to demonstrate signif-
icant differences in viral load, the selective pressure for se-
quence change to restore MHC-downregulating activity clearly
indicates that the absence of this activity did serve to limit the
replication of the virus at least somewhat.
A reasonable portion of the nef clones at 16 weeks lost the
ability to downregulate CD28, but by 32 weeks the vast major-
ity of clones behaved like the wild type for both CD28 and
MHC downregulation. This result suggests that some of the
sequence evolution between 16 and 32 weeks resulted in res-
toration of CD28-downregulating activity and further suggests
that downregulation of CD28 is also a biologically relevant
functional activity of Nef.
The large number of functional activities assigned to Nef has
raised doubts about whether all are relevant. Our results val-
idate MHC downregulation as a biologically relevant func-
tional activity of Nef. It should be possible to use the SIV/
rhesus monkey system as we have done here to validate the
relative importance of other functional activities of Nef. Also,
vaccine strategies that employ Nef expression as one compo-
nent of the immunogen should modify the nef gene in such a
way as to eliminate the ability to downregulate MHC class I.
We thank Hannah Sanford and Jackie Gillis for technical assistance
and John Altman for providing the SIV Gag181-189Mamu A*01 tet-
This work was supported by PHS grants RR00168 (NEPRC),
AI25328 (R.C.D.), AI35365 (R.C.D.), AI45314 (R.P.J.) and AI42561
(J.S.). This work was also funded in part with federal funds from the
National Cancer Institute, National Institutes of Health, under con-
tract NO1-CO-124000 (J.L.). T.S. is a Charles H. Revson Fellow.
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