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.
and development in transgenic mice expressing the HIV-1 nef gene. EMBO
50. Smith, B. L., B. W. Krushelnycky, D. Mochly-Rosen, and P. Berg. 1996. The
HIV nef protein associates with protein kinase C theta. J. Biol. Chem.
51. Spina, C. A., T. J. Kwoh, M. Y. Chowers, J. C. Guatelli, and D. D. Richman.
1994. The importance of nef in the induction of human immunodeficiency
virus type 1 replication from primary quiescent CD4 lymphocytes. J. Exp.
51a.Stevenson, P. G., J. S. May, X. G. Smith, S. Marques, H. Adler, U. H.
Koszinowski, J. P. Simas, and S. Efstathiou. 2003. K3-mediated evasion of
CD8(?) T cells aids amplification of a latent gamma-herpesvirus. Nat. Im-
52. Sugimoto, C., K. Tadakuma, I. Otani, T. Moritoyo, H. Akari, F. Ono, Y.
Yoshikawa, T. Sata, S. Izumo, and K. Mori. 2003. nef gene is required for
robust productive infection by simian immunodeficiency virus of T-cell-rich
paracortex in lymph nodes. J. Virol. 77:4169–4180.
53. Swigut, T., M. Greenberg, and J. Skowronski. 2003. Cooperative interactions
of simian immunodeficiency virus Nef, AP-2, and CD3-zeta mediate the
selective induction of T-cell receptor-CD3 endocytosis. J. Virol. 77:8116–
54. Swigut, T., A. J. Iafrate, J. Muench, F. Kirchhoff, and J. Skowronski. 2000.
Simian and human immunodeficiency virus Nef proteins use different sur-
faces to downregulate class I major histocompatibility complex antigen ex-
pression. J. Virol. 74:5691–5701.
55. Swigut, T., N. Shohdy, and J. Skowronski. 2001. Mechanism for down-
regulation of CD28 by Nef. EMBO J. 20:1593–1604.
56. Swingler, S., B. Brichacek, J. M. Jacque, C. Ulich, J. Zhou, and M. Steven-
son. 2003. HIV-1 Nef intersects the macrophage CD40L signalling pathway
to promote resting-cell infection. Nature 424:213–219.
57. Swingler, S., A. Mann, J. Jacque, B. Brichacek, V. G. Sasseville, K. Williams,
A. A. Lackner, E. N. Janoff, R. Wang, D. Fisher, and M. Stevenson. 1999.
HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected mac-
rophages. Nat. Med. 5:997–1103.
58. Wang, J. K., E. Kiyokawa, E. Verdin, and D. Trono. 2000. The Nef protein
of HIV-1 associates with rafts and primes T cells for activation. Proc. Natl.
Acad. Sci. USA 97:394–399.
59. Wolf, D., V. Witte, B. Laffert, K. Blume, E. Stromer, S. Trapp, P. d’Aloja, A.
Schurmann, and A. S. Baur. 2001. HIV-1 Nef associated PAK and PI3-
kinases stimulate Akt-independent Bad-phosphorylation to induce anti-
apoptotic signals. Nat. Med. 7:1217–1224.
60. Xu, X. N., B. Laffert, G. R. Screaton, M. Kraft, D. Wolf, W. Kolanus, J.
Mongkolsapay, A. J. McMichael, and A. S. Baur. 1999. Induction of Fas
ligand expression by HIV involves the interaction of Nef with the T-cell
receptor zeta chain. J. Exp. Med. 189:1489–1496.
61. Xu, X. N., G. R. Screaton, F. M. Gotch, T. Dong, R. Tan, N. Almond, B.
Walker, R. Stebbings, K. Kent, S. Nagata, J. E. Stott, and A. J. McMichael.
1997. Evasion of cytotoxic T lymphocyte (CTL) responses by Nef-dependent
induction of Fas ligand (CD95L) expression on simian immunodeficiency
virus-infected cells. J. Exp Med. 186:7–16.
62. Yang, O. O., P. T. Nguyen, S. A. Kalams, T. Dorfman, H. G. Gottlinger, S.
Stewart, I. S. Chen, S. Threlkeld, and B. D. Walker. 2002. Nef-mediated
resistance of human immunodeficiency virus type 1 to antiviral cytotoxic T
lymphocytes. J. Virol. 76:1626–1631.
63. York, I. A., C. Roop, D. W. Andrews, S. R. Riddell, F. L. Graham, and D. C.
Johnson. 1994. A cytosolic herpes simplex virus protein inhibits antigen
presentation to CD8? T lymphocytes. Cell 77:525–535.
13344 SWIGUT ET AL.J. VIROL.