NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001 65

ARTICLES

Infection with HIV-1 usually requires binding of viral-envelopeglycoprotein (gp)120 to the primary receptor CD4 expressed onhelper T cells and monocytes/macrophages1. CD8+ cells have animportant protective role against HIV-1. Although previousstudies have demonstrated that HIV-1 can occasionally infectCD8+ T cells, infection of CD8+ cells was thought to be mediatedthrough CD4 receptors expressed either transiently in CD8+ cellsor in double-positive T cells2,3. We have previously reported iso-lation of multiple CD8+ T-cell clones from two AIDS patients thatspontaneously produced HIV-1 (refs. 4,5). Here we study twoHIV-1 isolates, AD3.v6 and AD3.v22, that were produced by in-fected CD8+ clones from an AIDS patient, AD3 (ref. 4). BecauseCD8+ cells produced these viruses, we investigated whether theseviruses were capable of infecting CD8+ cells in vitro.

Primary CD8+ lymphocytes as targetsWe used phytohemagglutinin (PHA)-activated total peripheralblood lymphocytes (PBL) and a purified population of CD4+ orCD8+ cells from normal donors to compare the replication kinet-ics of AD3.v6 and AD3.v22 viruses. These viruses replicated inPBL and CD4+ cells (Fig. 1a and b). In repeated experiments,these viruses were also able to replicate in CD8+ cells to a levelthat matched that of CD4+ cells, indicating that CD8+ lympho-cytes may be targets for these viruses (Fig. 1a and b). As a control,we used CD4-tropic HIV-1 IIIB (IIIB) viruses that primarily repli-cated in PBL and CD4+ cells but not in CD8+ cells (data notshown).

Several lines of evidence indicated that CD4+ cells had no rolein virus production by the CD8+ population. First, we moni-tored phenotypes of purified cells throughout the experiments.A small number of cells in the CD8+ population were CD4+ be-fore infection (0 day) (Fig. 1c), at the time of peak virus produc-tion (18 day) (Fig. 1c) or at time-points in between (data not

shown) indicating that viruses were probably produced mostlyby CD4– cells. Second, compared with purified CD8+ cells, PBLcontained a much larger number of CD4+ cells (Fig. 1c). HadCD4+ cells been the only targets for these viruses, virus produc-tion would have been higher in PBL compared with CD8+ cells.Third, although these viruses were able to replicate in CD4+

cells, virus production in CD8+ cells was comparable with thatof CD4+ cells (Fig. 1a and b) indicating that CD8+ cells areequally good targets as CD4+ cells. Further evidence that conta-minating CD4+ cells did not produce viruses in purified CD8+

cells comes from triple-color FACS analyses. With AD3.v6-in-fected CD8+ cells, at eight days post infection (8 dpi) whenvirus-expressing cells could be detected for the first time, most(72.1%) virus-expressing cells were CD8+/CD4– (Fig. 1d). At thisstage, virus production in CD8+ cells was not detected by ELISA(Fig. 1a) ruling out the possibility of CD4 downmodulation.Moreover, for triple-color analyses, we fixed cells prior to stain-ing with antibodies (see Methods). Therefore, intracellular (ordownmodulated) antigens are likely to be detected by suchanalyses. Thus, virus production in CD8+ cells did not involveCD4 expression. The number of CD8+/CD4– cells expressingHIV-1 increased with progression of infection, and around thetime of peak virus production (19 dpi), all HIV-expressing cellswere CD8+/CD4– (Fig. 1d), indicating that single CD8+ cells pre-sent in purified CD8+ population were primary targets for theseviruses. Also, in experiments where we removed both CD4+ andCD8+ cells from PBL and residual (non-CD4/CD8) cells were in-fected, we detected little or no virus production, indicating thatCD8–/CD4– cells present in CD8+ population might not be thetargets for these viruses (data not shown). Expression of CD8was downmodulated after infection of CD8+ cells with AD3.v6(Fig. 1e) or AD3.v22 (Fig. 1f) viruses. CD4 was also downmodu-lated after infection of CD4+ cells with these viruses (data not

Isolation of primary HIV-1 that target CD8+ T Lymphocytesusing CD8 as a receptor

KUNAL SAHA1.2, JIANCHAO ZHANG1, ANIL GUPTA1, RAJNISH DAVE1, MERON YIMEN1 & BOUCHRA ZERHOUNI1

1Division of Molecular Medicine, Department of Pediatrics and 2Department of Molecular Virology,Immunology and Medical Genetics, Children’s Research Institute and

The Ohio State University Medical Center, Columbus, Ohio 43205, USA Correspondence should be addressed to K.S.; email: sahak@pediatrics.ohio-state.edu

HIV-1 use CD4 receptors to infect their primary targets, CD4+ cells, whereas CD8+ cells have aprotective role against HIV-1. We recently isolated HIV-1-producing CD8+ clones from two AIDSpatients. Here we show that although HIV-1 produced by CD8+ cells maintained the ability to in-fect CD4+ cells, these viruses were able to infect CD8+ cells independent of CD4. Evidence indi-cates that these viruses used CD8 as a receptor to infect CD8+ cells. First, expression of CD8 wasdownmodulated after infection. Second, anti-CD8 antibodies blocked viral entry and replicationin CD8+ cells. Finally, resistant cells became susceptible after expression of CD8. Although theseviruses used CXCR4 to enter CD4+ cells, it seems that infection of CD8+ cells was independent ofCXCR4 or CCR5 co-receptors. Novel changes were observed in envelope sequences of CD8-tropicviruses. These results provide initial evidence that HIV-1 can mutate to infect CD8+ cells usingCD8 as a receptor.

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

Fig. 2 a, Expression of CD8, CD4 and HIV-1 mRNA in CD8+/CD4– popu-lation. Cells were obtained by sorting uninfected or 6 days post-infection.Lanes 2, 6, 10 are infected with AD3.v6 and lanes 3, 7, 11 are infectedwith AD3.v22 viruses. Lanes 4, 8,12 are uninfected cells. RT-PCR amplifi-cation of CD8 (lanes 2–5), CD4 (lanes 6–9) and HIV-1 (gag) (lanes 10–13)were performed as described. CD4+ 8E51 cells were used as positive con-trols for HIV-1 (lane 13), CD4 (lane 9) and used as negative control forCD8 in lane 5. Lane 14 is water control for PCR contamination. Each sam-ple was amplified with or without RT to rule out DNA contamination (data

66 NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001

ARTICLES

shown) indicating maintenance of CD4 tropism.Some CD8+ cells co-express CD4 molecules after stimula-

tion in vitro (Fig. 1c). AD3.v6 and AD3.v22 viruses may possi-bly have infected double-positive cells present in CD8+

population using CD4 receptors, and CD4 expression wasdownmodulated after infection, which may give rise to phe-notypically single CD8+ cells. Alternatively, CD8+ cells maypossibly express very low but undetectable (by FACS) levels ofCD4, which these viruses may have used to enter CD8+ cells.To exclude such possibilities, we sorted purified CD8+ cellsshortly after infection and tested these stringently selected

CD8+ cells for expression of CD4 and HIV-1 by sensitive RT-PCR. AD3.v6- and AD3.v22-infected and sorted CD8+ cells ex-pressed CD8 and HIV-1 (gag) but no CD4, whereas uninfectedCD8-positive cells expressed CD8 but no CD4 or HIV-1 (Fig.2a). Similarly sorted CD8+ cells after infection with CD4-tropic (JR-FL) viruses did not express HIV-1 or CD4 (data notshown). Our method of detection of CD4 expression by RT-PCR is highly sensitive and can detect as low as 1 CD4+ cell in100,000 cells (data not shown). Thus, sorted CD8+ cells wereclearly free from CD4 contamination. These data establishthat CD4 was not involved in virus production by CD8+ cells.

� � �

�

� � � ��

�

� � ��

�

� �

0 3 7 11 14 17 20 240

100

200

300

400

500

600

700

800

900

1000

days

�� � ����

p24

(n

g/m

l)

� �

��

�

� �� �

�

�

�

�� � ��

��

�

�

0 3 7 11 14 17 20 240

50

100

150

200

250

300

days

� ��

p24

(n

g/m

l)

.

7

5

3

1

0 .

7

5

3

1

0

CD8-PE

PBL control (0 day)

CD4+ control (0 day) CD4-AD3.v6 (18 day) CD8-AD3.v6 (18 day)

CD8-AD3.v22 (18 day)

CD4

CD

8

CD8+ control (0 day) CD4-AD3.v22 (18 day)

c

.1 1 10 100 10000

.1

1

10

1

00

1

00

0

.1 1 10 100 1000

.1 1 10 100 1000

CD4+ control (18 day)

.1

1

10

1

00

1

00

0

CD8+ control (18 day)

.1

1

10

1

00

1

00

0

.1 1 10 100 1000

.1

1

10

1

00

1

00

0

.1 1 10 100 1000

.1

1

10

1

00

1

00

0

.1 1 10 100 1000.1

1

10

1

00

1

00

0

.1 1 10 100 1000

.1

1

10

1

00

1

00

0

.1 1 10 100 1000.1

1

10

1

00

1

00

0

.1 1 10 100 1000

.1

1

10

1

00

1

00

0

CD8-PE

p2

4-F

ITC

CD4-PC5

CD

8-P

E

8dpi

8dpi

15dpi

15dpi

Uninfected

19dpi

19dpi

0% 1.75% 2.35 % 1.39%

72.1 % 80.1% 99.6 %

Fig. 1 a and b, Infection of PBL, purified CD8+ and CD4+ cells with AD3.v6 (a)and AD3.v22 (b) viruses. PBL ( ), CD4+ ( ) or CD8+ ( ) cells were infectedwith AD3.v6 or AD3.v22 viruses and virus production was detected at regularintervals. c, Analysis of CD4 and CD8 expression before or after infection withAD3.v6 and AD3.v22 viruses. Cells were infected as in (a and b) above and an-alyzed by double-color FACS analysis5 before infection (0 day), at regular inter-vals after infection (data not shown) and at the time of peak virus production(18 day). d, Expression of CD4 and HIV-1 (p24) by purified CD8+ cells after in-fection with AD3.v6 viruses. Uninfected and infected cells were analyzed atregular intervals by triple-color FACS. CD8+ cells expressing p24 antigens (cir-

cled in upper panel) were gated and examined for expression of CD4 (lowerpanel). e and f Down-modulation of CD8 expression in CD8+ cells after infec-tion with AD3.v6 (e) or AD3.v22 (f) viruses. CD8 expression in the infected cells(11 dpi) was measured by FACS using PE-labeled anti-CD8 antibodies (Sigma)and compared with uninfected control cells; AD3.v6/v22-infected (yellowlines), uninfected (orange lines). g and h, Induction of syncitia in PBL byAD3.v22 viruses and not by AD3.v6 viruses. PBLs were infected and 3 daysafter infection, syncitia were seen with AD3.v22 (g), but not with AD3.v6 (h),viruses. Arrows point to some of the syncitia.

���

a b

c

d

e f

g

h

1 2 3 4 5 6 7 8 9 10 11 12 13 14

CD8 CD4

gag

a

�

�

�

��

�

�

�

�

��� � � � �

0 1 4 7 10 130

1

2

3

4

5

6

7

Days

� �

p24

(n

gm

l)

a b

not shown). b, Virus production by sorted [as in (2a)] CD8+/CD4– orCD8–/CD4– cells. Equal numbers (100,000) of infected and sorted cellswere plated and virus production measured at regular intervals.CD8+/CD4– cells infected with AD3.v6 (�) or AD3.v22 �) viruses andCD8-/CD4-cells infected with AD3.v6 (�) or AD3.v22 (�) viruses areshown.

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001 67

ARTICLES

Further evidence that viruses produced by CD8+ cells were in-deed released by CD8+/CD4– cells come from culture of thesesorted cells. When sorted CD8+/CD4– or CD8–/CD4– cells fromCD8+ population were cultured, we detected virus productionprimarily from CD8+/CD4– cells (Fig. 2b). Together, these re-sults demonstrate that AD3.v6 and AD3.v22 viruses are ableto target CD8+/CD4– cells and CD4 played no role in infectionof CD8+ cells. Finally, AD3.v22 (Fig. 1g), but not AD3.v6 (Fig.1h) viruses were able to form discrete syncitia in PBL indicat-ing a phenotypic difference between these two isolates,though the significance of this difference remains unclear.

Infection of CD8+ T-cell lineExperiments with primary CD8+ cells strongly indicate thatCD8+ cells may be targets for AD3.v6 and AD3.v22 viruses.But because primary cells contain a mixture of different celltypes, to further establish CD8 tropism for these viruses, weused a CD8+ T-cell line, KRCD8 (ref. 6). AD3.v6 and AD3.v22,but not IIIB, viruses were able to infect KRCD8 cells (Fig. 3a).We detected no expression of CD4 in KRCD8 cells whetheruninfected (Fig. 3b) or after infection (data not shown). Evenby sensitive RT-PCR, no CD4 mRNA was detected in KRCD8cells before or after infection (Fig. 3c). These results show thatKRCD8 cells were infected through a CD4-independent mech-anism. Previous reports of CD4-independent infection withHIV have implicated CCR5 or CXCR4 as the alternative path-way for infections7–10. Uninfected KRCD8 cells did not expressCCR5 (Fig. 3d), CXCR4 (Fig. 3f) nor did infected KRCD8 cells(data not shown) indicating that infection of KRCD8 cellsprobably was independent of CCR5 or CXCR4. Together,these studies confirm that AD3.v6 and AD3.v22 viruses areable to infect CD8+ cells in a CD4-independent manner.However, these CD8-tropic viruses have maintained un-changed ability to infect CD4+ cells.

CD8 as a receptorBecause CD8 expression was downmodulated in AD3.v6- andAD3.v22-infected cells, we investigated whether these virusesused CD8 as receptors. To investigate the role of CD8 as a recep-tor, we used HeLa T8+ cells, a CD8-transfected tumor cell linethat does not express CD4. HeLa T8+ cells are generally used asnegative controls in experiments with HeLa T4+ cells that are sus-ceptible to CD4-tropic HIV-1. We infected HeLa T8+, HeLa T4+ orparental HeLa cells with AD3.v6, AD3.v22 or IIIB viruses. We ob-served viral replication in HeLa T8+ cells between 7 and 15 daysafter infection with AD3.v6 and AD3.v22, but not with IIIB,viruses (Fig. 4a). All three viruses, however, were able to infectHeLa T4+, but not parental HeLa cells (data not shown). LikeKRCD8 cells, infection of HeLa T8+ cells with AD3.v6 andAD3.v22 viruses was independent of CD4 expression as moni-tored through RT-PCR (data not shown). Expression of CD8 wasalso downmodulated in HeLa T8+ cells after infection withAD3.v6 and AD3.v22 viruses (data not shown) indicating therole of CD8 as a receptor.

Infection of HeLa T8+ cells with AD3.v6 and AD3.v22 virusesresulted from co-culture with infected PBL. The chances for pos-sible contamination with CD4+ cells present in PBL in such sce-narios are small because co-cultures with HeLa cells did notproduce any virus. We nevertheless performed further experi-ments using cell-free viruses. In agreement with co-culture ex-periments, AD3.v6 (Fig. 4b) and AD3.v22 (data not shown)viruses were able to infect HeLa T8+ and HeLa T4+, but not HeLa,cells whereas IIIB viruses infected only HeLa T4+, but not HeLaT8+ or HeLa cells. These results demonstrate that AD3.v6 andAD3.v22 viruses used CD8 molecules to infect HeLa T8+ cells.

To further substantiate the role of CD8 as a receptor we in-fected, HeLa T8+ cells with AD3.v6, AD3.v22 or IIIB viruses in thepresence of anti-CD8 (C1), anti-CD4 or isotype control antibod-ies. AD3.v22 viruses were able to infect HeLa T8+ and HeLa T4+

� �

�

�

�

�

�

� � � �0 3 7 10 14

0

0.5

1

1.5

2

2.5

3

3.5

Days

p24

(ng/

ml)

� �

CD

8-P

E

CD4-FITC

1000 100 10 1.1

75

56

37

18

0

7556

3718

0

1000 100 10 1.1

75

56

37

18

0

75

56

37

18

0

1000 100 10 1.1 1000 100 10 1.1

75

56

37

18

0

CD4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

CD8

Fig. 3 a, Infection of KRCD8 cells by AD3.v6 and AD3.v22 viruses. Cellswere infected with AD3.v6 (�), AD3.v22 (�) or HIV-1/IIIB (�) virusesand p24 production was measured at regular intervals. b, Expression ofCD4 or CD8 in KRCD8 cells was tested by double-color FACS analysis. c,Expression of CD4 and CD8 mRNA by RT-PCR in KRCD8 cells before andafter infection with AD3.v6 and AD3.v22 viruses. RT-PCR for CD4 andCD8 was performed as in (2a). Lane 1: M.W.; Lanes 2–8 are with CD8primers. Lane 2, uninfected cells (2d); lane 3, AD3.v6-infected (2 dpi);lane 4, AD3.v22-infected (2 dpi); lane 5, uninfected cells (6d); lane 6,AD3.v6-infected (6 dpi); lane 7, AD3.v22-infected (6 dpi); lane 8, CD8positive control. Lanes 9–14 are identical samples as in lanes 2–7, re-spectively, but with CD4 primers. Lane 15 is positive control for CD4and lane 16 is water control for PCR contamination. Each sample wastested negative for DNA contamination (data not shown). d–g,Expression of CCR5 or CXCR4 in KRCD8 cells. KRCD8 cells were stainedindirectly with FITC-labeled anti-CCR5 (d) and anti-CXCR4 (f) or with

isotype control antibodies and analyzed by FACS. PHA-activated PBMC(e) and MT-2 cells (g) were used as positive control cells for CCR5 andCXCR4, respectively. Control antibodies (yellow lines), anti-CCR5/CZCR4 (orange lines).

a b c

d e

f g

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

gag

1 2 3 7 188 9 104 5 6 11 12 13 14 1516 17

68 NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001

ARTICLES

(Fig. 4c), but not HeLa cells (data not shown), and viral entryinto HeLa T8+ cells was blocked with anti-CD8, but not with iso-type control or antibodies against CD4 (anti-CD4). In contrast,viral entry into HeLa T4+ cells was prevented by anti-CD4, butnot with isotype control or antibodies against CD8 (anti-CD8).As expected, IIIB viruses were only able to infect HeLa T4+, butnot HeLa T8+ or HeLa cells. Infection with AD3.v6 viruses wasidentical to that of AD3.v22 viruses (data not shown). Thesestudies demonstrate that AD3.v6 and AD3.v22 viruses infectedHeLa T8+ cells using CD8 receptors and HeLa T4+ cells using CD4receptors. To further test the role of CD8 as a receptor, we gener-ated a monkey kidney cell line (COS-T8) that constitutively ex-pressed high levels of human CD8 (data not shown). As withHeLa T8+ cells, AD3.v6 (Fig. 4d) and AD3.v22 (data not shown)viruses were able to infect COS-T8 but not parental COS cells ,

whereas IIIB viruses failed to infect either of these cell lines. Wedetected no CD4 expression in COS-T8 cells either before or afterinfection (data not shown). These results establish that AD3.v6and AD3.v22 viruses can infect CD8+ cells using CD8 as a recep-tor.

As KRCD8 cells were susceptible to AD3.v6 and AD3.v22viruses, we investigated whether these viruses also used CD8 re-ceptors to infect these lymphoid cells. We used AD3.v6, AD3.v22or IIIB viruses to infect KRCD8 cells or MT-2 cells (as control) inthe presence of anti-CD8, -CD4 or isotype control antibodies.Whereas anti-CD4 or isotype control antibodies had no effect oninfection of AD3.v6 or AD3.v22 viruses, anti-CD8 (C1) blockedentry of these viruses into KRCD8 cells (Fig. 5a). Anti-CD8, how-ever, were not able to prevent infection of CD4+ MT-2 cells byAD3.v6 (data not shown), AD3.v22 or IIIB viruses (Fig. 5b). In

gag

1 2 3 4 5 6 7 8 9 10 11 12

gag

1 2 3 4 5 6 7 8 9 10 11 12Fig. 4 a, Infection of HeLa T8+ cells with AD3.v6 and AD3.v22 viruses. HeLaT8+ cells were infected with AD3.v6 (�) and AD3.v22 (�) by co-culture with in-fected PBL and virus production (p24) was followed. b, Infection of HeLa T8+

cells with cell-free AD3.v6 or IIIB viruses. HeLa T8+, HeLa T4+ or HeLa cells wereinfected with AD3.v6 or IIIB (as control) viruses as described and infection wasdetected by PCR at 7 dpi. Lanes 2–4 are infected with AD3.v6, lanes 5–7 are in-fected with IIIB viruses. Lanes 2, 5, 8 are HeLa T8+ cells; lanes 3, 6, 9 are HeLa T4+

cells and lanes 4, 7, 10 are HeLa cells. Lanes 8–10 are uninfected cells only; lane11 is positive control cells (8E51) and lane 12 is water control for PCR contami-nation. c, Blocking of entry of AD3.v22 viruses into HeLa T8+ cells by anti-CD8antibodies. HeLa T8+ and HeLa T4+ (as control) cells were treated with differentantibodies infected with DNAse-treated viruses and viral entry detected by PCRas described. Lanes 2–8, 12, 13 are infected with AD3.v22 viruses and lanes9–11, 14 are infected with IIIB viruses (as control). Lanes 2–4, 9, 12, 15 are HeLaT8+ cells; lanes 5–7, 10, 13, 14, 16 are HeLa T4+ cells and lanes 8, 11 are HeLacells. Lanes 2, 5 are with isotype control antibodies; lanes 3, 6 are with anti-CD8antibodies (C1); lanes 4, 7 are with anti-CD4 antibodies; Lanes 12–14 are

treated and lysed at 4 °C as controls for contaminating viral DNA. Lanes 15, 16are uninfected cells only. Lane 17 is positive control (8E51) and lane 18 is watercontrol for PCR contamination. d, Infection of COS T8 cells with AD3.v6 viruses.DNAse-treated AD3.v6 or IIIB (as control) viruses were used to infect COS orCOS-T8 cells and viral entry was detected by PCR. Lanes 2–4 are COS cells; lanes5–7 are COS-T8 cells and lanes 8–10 are HeLa T4+ cells. Lanes 3, 6, 9 are in-fected with AD3.v6 and lanes 4, 7, 10 are infected with IIIB viruses. Lanes 2, 5, 8are uninfected cells. Lane 11 is 8E51cells (positive control) and lane 12 is watercontrol for PCR contamination.

�

� �

�

0 4 7 15 180

2

4

6

8

Days

� �

�

�

��

p24

(ng

/ml)

a

c

d

b

gag

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1717

gag

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1515

Fig. 5 (a) Blocking of entry of AD3.v6 and AD3.v22 viruses into KRCD8cells by anti-CD8 antibodies. Cells were treated with anti-CD8, -CD4 orisotype control antibodies infected with Dnase-treated viruses and viralentry was detected by PCR as described in Methods. Lanes 2-6 and 14are infection with AD3.v6; lanes 7-11 and 13 are infected with AD3.v22viruses. Isotype control antibodies (lanes 2,7); anti-CD8 antibody, SPV-T8 (lanes 3,8); anti-CD8 antibody,C1 (lanes 4, 9); anti-CD8 antibody, C2(lanes 5, 10); anti-CD4 antibody (lanes 6, 11). Lane 12 is uninfected cellsonly. Lanes 13 and 14 are cells infected and lysed at 4°C to exclude viralDNA contamination. Lanes 15 and 16 are water controls for PCR conta-mination. Lane 17 (8E51) is positive control. (b) Blocking of entry ofAD3.v22 and IIIB viruses into MT-2 cells by anti-CD4 antibodies. Cellswere treated with anti-CD8, anti-CD4 or isotype control antibodies, in-fected with DNAse-treated viruses and viral entry was detected by PCR asdescribed in Methods. Lanes 2-5 and 12 are infected with AD3.v22viruses; lanes 6-9 and 13 are infected with IIIB viruses. Isotype control an-

tibodies (lanes 2, 6); anti-CD8 antibody, C1 (lanes 3, 7); anti-CD8 anti-body, C2 (lanes 4, 8); anti-CD4 antibody (lanes 5, 9). Lane 10 is unin-fected cells only. Lanes 11-13 are cells infected and lysed at 4°C to ruleout input viral DNA contamination. Lane 14 is positive control (8E51).Lane 15 is water control for PCR contamination.

a

b

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001 69

ARTICLES

contrast, anti-CD4 blocked infection of MT-2 cells by AD3.v6(data not shown), AD3.v22 or IIIB viruses (Fig. 5b). Two othermonoclonal antibodies against CD8 also failed to prevent infec-tion of KRCD8 cells by AD3.v6 and AD3.v22 viruses (Fig. 5a).Together, these studies confirm that AD3.v6 and AD3.v22viruses used CD8 receptors for infection of CD8+ cells. These re-sults also demonstrate that these viruses used CD4 receptors toinfect CD4+ cells.

We further tested the ability of anti-CD8 to inhibit replica-tion of CD8-tropic viruses in primary CD8+ cells. Replicationof AD3.v6 (Fig. 6a) and AD3.v22 (Fig. 6b) viruses was signifi-cantly inhibited by anti-CD8 (C1 and C2), but not by isotypecontrol antibodies. We did not detect increased virus produc-tion after discontinuing the addition of anti-CD8 after thefirst two weeks (data not shown). Such results could not be at-tributed to the difference in proliferation of the cells in thepresence of these antibodies. However, anti-CD8 had no effecton replication of these as well as IIIB viruses in CD4+ cells(data not shown). It is not clear why anti-CD8 (C2) inhibitedreplication in primary CD8+ cells but was not able to blockvirus entry into KRCD8 cells (Fig. 5a). It is possible that thisantibody blocked viral entry only partially, thereby sensitivePCR was able to detect viral signals. CD8-tropic viruses main-tained unchanged ability to use CD4 receptors as well andshould be considered as dual (CD4/CD8)-tropic. As CD4 is co-expressed in some CD8+ cells, these viruses conceivably mayuse CD8 as well as CD4 receptors to infect double-positivecells. In agreement with this notion, even though anti-CD4failed to prevent infection of KRCD8 (Fig. 5a) or HeLa T8+ (Fig.4c) cells that expressed no CD4, these antibodies were able toinhibit replication of AD3.v6 and AD3.v22 viruses, albeit onlypartially, in primary CD8+ cells (data not shown).Nevertheless, several lines of evidence including the ability ofAD3.v6 and AD3.v22 viruses to infect CD8-transfected cells

(Fig. 4b and d), their susceptibility to viral entry (Figs. 4c and5a) and replication (Fig. 6a and b) by anti-CD8, and theirdownmodulation of CD8 expression (Fig. 1e and f) establishthat these viruses used CD8 as a receptor to infect CD8+ cells.

Novel changes in envelopesCellular tropism of HIV-1 is primarily determined by viral en-velopes1. In order to further characterize CD8-tropic HIV-1, wesequenced the envelopes of AD3.v6 and AD3.v22 viruses. Wenoticed extensive changes all over gp120 in these viruseswhen compared with HXB2 (Fig. 7), a prototype CD4-tropicisolate. Although AD3.v6 and AD3.v22 viruses belong to CladeB, phylogenetic analyses showed a monophyletic clustering ofthese sequences (data not shown). When compared with pub-lished sequences, we found the nearest relative of these viruses(WEAU1.6) to be substantially different. The changes weremost extensive in variable (V) loops and a striking feature ofthese changes was an extended V1–V2 loop. Extensive differ-ences were also present in the important V3 loop when com-pared with its closest match. Only a single point change was

B�

� � � �

� � �0 3 7 11 14 17 20

0

50

100

150

200

250

days

� � �

�

�

�

p24 (

ng

/ml)

� � �

�

�

��

� � � �0 3 7 11 14 17 20

days

� �

� �

Fig. 6 a and b, Inhibition of infection of CD8+ cells with AD3.v6 andAD3.v22 viruses by anti-CD8 antibodies. Primary CD8+ lymphocytes weretreated with anti-CD8 antibodies (C1 and C2) or isotype control antibodiesand infected with AD3.v6 (a) and AD3.v22 (b) viruses as described. Virusproduction was monitored by harvesting culture supernatants at regular in-tervals. Control antibody (�), C1 anti-CD8 antibody (�), C2 anti-CD8 anti-body (�).

Fig. 7 Sequence analysis of envelopes of AD3.v6 and AD3.v22 viruses incomparison to HXB2 and WEAU1.6, closest matched isolate to AD3.v6and AD3.v22 viruses. Dash (–) signifies 100% identity among all isolatesand dot (.) signifies absence/deletion of a base. To highlight the differ-ences between WEAU1.6 and AD3.v6/AD3.v22 viruses, the differenceshave been colored in red with WEAU1.6 sequences. Other coloredboxes are to highlight important sequences and have been discussed inthe text. The two envelope sequences reported here have been submit-ted to GenBank (accession number AF219627 for AD3.v6 andAF219628 for AD3.v22).

a b

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

70 NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001

ARTICLES

observed in the entire gp41 between AD3.v6 and AD3.v22viruses (data not shown). The CD4 binding region (in blue)and other residues that are important for CD4 tropism (greenboxes) remained unchanged in these viruses indicating thatextensive mutations in AD3.v6 and AD3.v22 viruses wereprobably not random events.

Infection of CD8+ cells is independent of CCR5 and CXCR4AD3.v6 and AD3.v22 viruses form syncitia in MT-2 cells4 indicat-ing that these viruses probably use CXCR4 co-receptors to infectCD4+ T-cells. Additionally, using cell lines that expressed differ-ent co-receptors in CD4+ (U87-CD4) cells, these viruses havebeen found to use CXCR4 (J. Mullins, pers. comm.). Thus, theseviruses evidently use CXCR4 co-receptors in the context of CD4receptors. KRCD8 cells that did not express CCR5 or CXCR4,however, were nonetheless susceptible to AD3.v6 and AD3.v22viruses (Fig. 3a). Additionally, antibodies against CCR5 orCXCR4 were unable to inhibit infection of CD8+ cells by theseviruses (data not shown). Moreover, these viruses were also ableto infect a monkey cell line (COS T8) (Fig. 4d) that did not ex-press CXCR4 or CCR5 (data not shown). Thus, it appears thatthese co-receptors are not involved in infection through CD8 re-ceptors.

Although no sequence can be definitively correlated with aspecific HIV-1 phenotype, critical residues in the envelope havebeen identified that are important for cellular tropism throughCCR5 or CXCR4. Using chimeric HXB2 viruses, Hung et al.11

have demonstrated that in the context of IGX motif (position348–350 in Fig. 7) in the V3 loop and with a basic residue at po-sition 331, viruses are incapable of using CCR5. These residues(red boxes) were identical among AD3.v6, AD3.v22 or HXB2viruses that cannot use CCR5 indicating that AD3.v6 andAD3.v22 viruses probably do not use CCR5 co-receptors. Also,infection of CD4-negative cells through CCR5 was correlatedwith specific changes (pink boxes) resulting in loss of N-linkedglycosylation sites at distinct regions10. Although extensivechanges in the V1-V2 loop of AD3.v6 and AD3.v22 viruses wereobserved, these viruses maintained the specific glycosylationsites (marked with (�) in Fig. 7). CD4-independent entrythrough enhanced use of CXCR4 has also been correlated withseven specific mutations (violet boxes) in NDK mutants9. A spe-cific T to S substitution (position 223 in fig. 7), not present in ei-ther AD3.v6 or AD3.v22 viruses, was necessary forCD4-independent infection by NDK mutants. Together, theseresults indicate that although AD3.v6 and AD3.v22 viruses useCXCR4 co-receptors for infection of CD4+ cells, these co-recep-tors may not be necessary for infection through CD8 receptors.We cannot, however, rule out the possibility of other unidenti-fied co-receptors involved in infection of CD8+ cells.

DiscussionHIV has evolved strategies to enable virus replication to con-tinue in spite of a strong immune response12,13. Evolution in vivoof a quasispecies of HIV that is adapted to use additional co-re-ceptors under selective immune pressure is well-established14.Here we show for the first time that HIV-1 can mutate to a formthat can target CD8+ cells. We further demonstrate that thesemutants used CD8 as a receptor to infect CD8+ cells. CD8-tropicviruses, however, still maintain the ability to infect CD4+ cellsusing CD4 receptors. Even though these viruses use CXCR4 co-receptors to infect CD4+ cells, it appears that these co-receptorshave no role for infection through CD8 receptors. Despite a

strong CD8+ cell-mediated immune response after primary HIVinfection, host defense eventually fails leading to the develop-ment of AIDS. Although several hypotheses including anergy,apoptosis and antigenic stimulation have been suggested15, theexact reason for ultimate failure of CD8+ cells remains unclear.Our findings, albeit from a single patient, provide a new hypoth-esis that may explain the failure of CD8+ cells in AIDS patients.With evolution of CD8-tropic HIV, productive infection of CD8+

cells may lead to functional defects or death in these cells lead-ing to a quantitative failure.

One might wonder why CD8-tropic HIV-1 were not identifieduntil now. It is possible that CD8-tropic viruses exist at a muchlower frequency compared with common CD4-tropic HIV-1.Primary HIV-1 is traditionally isolated by co-culture with PBLsfrom healthy donors that contain large numbers of CD4+ cellsthat may enable CD4-tropic viruses to replicate rapidly and pre-dominate in culture. The novel CD8-tropic viruses in this studywere isolated using a strategy that enhanced the chances for out-growth of CD8-tropic viruses. These viruses were generated frominfected CD8+ cells that were immortalized and cloned within 3to 5 days after isolation in the presence of a protease inhibitor,thus providing little opportunity for CD4-tropic strains tospread4,5. Although these virus-producing clones were immortal-ized by herpesvirus saimiri (HVS), it is unlikely that other extra-neous factors may have influenced these viruses to infect CD8+

cells. HVS-immortalized clones have been shown to produce typ-ical CD4-tropic isolates6,16 and used for important functionalstudies17,18. Furthermore, we have isolated biological clones fromPBL of the same patient (AD3) and envelope sequences, as well asbiological phenotypes of some of these viruses closely matched(including CD8-tropism) to that of AD3.v6 or AD3.v22 viruses,ruling out the possibility of HVS influencing CD8 tropism (un-published observation).

Although the V3 loop has been a major determinant of celltropism11,19, several reports have implicated V1–V2 loops in influ-encing HIV-1 infection20–23. Recent crystallographic resolution ofgp120 has revealed that the β-sheet that serves as a major contactsite after binding of gp120 is made up largely of amino acidsfrom C4 domain and V1–V2 stem24,25. It is likely that addition ofa large number of amino acids in the V1 loop and changes inother V-loop areas are responsible for CD8-tropism of AD3.v6and AD3.v22 viruses. Shankarappa et al.26 have reported a basicamino acid substitution at position 319 of the V3 loop in a pro-gressor, which was the only X4-conferring mutation.Interestingly, both AD3.v6 and AD3.v22 viruses had a basicamino acid substitution at the identical position (position 345 infig. 7). Finally, there is evidence that HIV-2 envelope can bind toCD8 in human T cells27. AD3.v6 and AD3.v22 viruses have alsobeen found to bind to CD8 receptors (data not shown).

CD8-tropic viruses in this study were isolated from a single pa-tient with AIDS. It remains unclear at present whether suchviruses may exist in other HIV-infected people. As reported in arecent abstract, HIV-1 has been implicated to target CD8+ T cellsin 3 of 5 patients (B.A. Sahai, pers. comm.). Along with B andCD4+ cells, CD8+ cells are the other predominant cell type inblood and lymphoid organs, the primary sites for HIV replica-tion. It is tempting to speculate that with increasing selectivepressure from a declining pool of CD4+ cells, HIV-1 is able to mu-tate to a form that can target CD8+ cells. Host immunity may failto protect against HIV-1 with direct infection of immune-compe-tent CD8+ cells. However, one must be cautious in extrapolatingthe role of such possibilities in clinical infections until future

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001 71

ARTICLES

studies with larger cohorts are able to test this hypothesis.Nevertheless, evidence of primary HIV-1 that infect CD8+ T cellsusing CD8 as a receptor should have important ramificationsthat include not only the obvious ones related to AIDS patho-genesis but also truly sobering implications with respect to therange of mutability and tropism of HIV-1.

MethodsVirus isolation and infection of CD4+ and CD8+ cells. Generation ofAD3.v6 and AD3.v22 viruses has been described4–6. Isolation of PBL and pu-rified CD4+ or CD8+ cells using antibody-coated magnetic beads has beendescribed5. Two million PHA-activated (1 µg/ml) PBL or purified CD4+ andCD8+ cells were infected with different AD3.v6, AD3.v22 and CD4-tropicIIIB or JR-FL (AIDS Research and Reagent Program, NIH) for 2 h at 37 °Cusing 0.05 pg of p24 per cell. Cells were washed, adjusted to 106 cells/mland cultured in complete RPMI 1640 medium supplemented with humanrecombinant IL-2 (50 units/ml) (Life Technologies, Grand Island, NewYork). Culture supernatants were collected at regular intervals and stockedat –80 °C until assayed for p24 antigen using a kit (Coulter, Hialeah,Florida). Phenotyping was performed by FACS at regular intervals using di-rectly labeled anti-CD4, -CD8 (Sigma) or indirectly labeled anti-CCR5 andanti-CXCR4 antibodies (see below) with appropriate isotype and cell con-trols28. Triple-color FACS analyses were performed using a kit from Coulterby fixing the cells first, followed by staining with FITC-p24, CD8-PE andCD4-PC5. In some experiments, purified uninfected or infected (6dpi) CD8+

cells were FACS sorted after staining with CD8-PE and CD4-PC5 antibodiesusing a gate to isolate CD8+/CD4– and CD8–/CD4– population. Sorted cellswere washed several times and either tested immediately for expression ofCD4, CD8 or HIV-1 mRNA by RT-PCR or cultured for virus production.

Infections of KRCD8 cells were performed essentially as primary cells asdescribed above. Infections of HeLa T8+, HeLa T4+ or parental HeLa cells(AIDS Research and Reagent Program, NIH) were performed either by co-culturing with infected PBL or with cell-free viruses in the presence of poly-brene (2 µg/ml). After overnight coculture or infection for 4 h with cell-freeviruses, cells were thoroughly washed, trypsinized and re-plated with freshmedium. At 5–7-day intervals, cells were trypsinized and re-plated. Culturesupernatants were collected at regular intervals and assayed for p24. Fornon-productive infections, infection was detected at routine intervals byPCR. For some experiments, DNAse-treated viruses were used for infectionas discussed below.

To generate human CD8-expressing COS cells, T8pMV7 vector (AIDSResearch & Reagent Program) expressing CD8 was used to transfect COS-7L cells (GIBCO-BRL). Lipofectamine 2000 (GIBCO-BRL), a lipid transfectingreagent was used for transfection according to the manufacturer’s instruc-tion. 24 h post-transfection, cells were re-plated at 1:20 dilution and afteranother 24 h, cells were put in selection medium. Individual clones wereisolated and screened by FACS to select clones expressing CD8.

Detection of CD4, CD8 and HIV-1. Expression of CD4 and CD8 moleculesin different cell types was measured by FACS (ref. 29) as well as by sensitiveRT-PCR using CD4- and CD8-specific primers as described5,29. Briefly, totalRNA was extracted using Rneasy Mini Kit (Qiagen, Valencia, California).DNA-free RNA was reverse transcribed using Qiagen Omniscript RT systemand tested for expression of CD4, CD8, or HIV-1 using specific primers. Fortesting of viral DNA, infected or control cells were collected and extractedDNA was amplified by PCR.

Inhibition of viral entry and replication. Viral stocks were treated withDNAse (Life Technologies) at 4000 U/ml for 1 h at 37 °C. At the same time,500,000 target cells were incubated on ice with anti-CD8, anti-CD4 or iso-type control (IgG1) antibodies (5 µg/ml). Monoclonal antibodies used inthese experiments are: anti-CD8 antibodies, Clone SPV-T8 (ZymedLaboratories, San Francisco, California); Clone B9.11 or C.1(Coulter/Immunotech, Miami, Florida); Clone SFC121Thy2D3 or C.2(Coulter/Immunotech). Anti-CD4 antibody is clone RPA-T4 (ZymedLaboratories). Cells were infected with DNAse-treated viruses for 2 h andafter further washes, cells were re-suspended in culture medium to whichrespective antibodies were added. After 24 h culture at 37 °C, cells werelysed and collected for PCR. To rule out the possibility of any carryover viral

DNA, control cells were incubated with DNAse-treated viruses on ice for 2h, washed and lysed immediately and tested by PCR.

For inhibition of viral replication, specific monoclonal antibodies or iso-type control antibodies (5 µg/ml) were added to purified CD8+ cells for 30min at room temperature prior to infection. After infection for 1 h, cellswere washed and re-suspended in culture medium to which respectivemonoclonal antibodies were added. Culture supernatants were collected atregular intervals and assayed for p24. Half of the medium was replacedtwice per wk with fresh medium containing respective antibodies except insome experiments where antibodies were stopped after 2 wk. Along withthe anti-CD4 and anti-CD8 antibodies described above, other antibodiesused in these experiments are: anti-CCR5 antibody, 2D7 (PharmingenInternational, San Diego, California) obtained through AIDS Research andReference Reagent Program; anti-CXCR4 antibody, 12G5 (from J. Hoxiethrough AIDS Research and Reference Reagent Program); and isotype con-trol antibodies (Sigma).

Sequence analysis. Full length envelope coding regions were amplified bynested PCR from genomic DNA using outer (5´-CTGGAAG-CATCCAGGAAGTCAGCC-3´ and 5´-GTCCCCAGCGGAAAGTCCCTTGTA-3´) and inner (5´-GAGACAGTGGCAATGAGAGTGAAGG-3´ and5´-CTTTTTGACCACTTGCCACCCATCTT-3´) primers. Amplified PCR frag-ment (2.6 kb) was purified and sequenced from both DNA strands using aset of 16 primers slightly modified as necessary as described30 by cycle se-quencing on an ABI 377 DNA Sequencer. Sequence assembly and compar-isons were performed with Lasergene (DnaStar, Madison, Wisconsin) aswell as with NCBI Blast server.

AcknowledgmentsWe thank P.R. Johnson, C.M. Walker, J.I. Mullins, A. van’ Wout, P. Gupta, B.Graham and J. Zack for helpful suggestions and critical review of themanuscript; R. Munson for advice for sequencing; K. Howe for help with thephylogenetic analyses; C. McAllister for assistance with flow cytometry; A.Dutcher for support in manuscript preparation; and the Children’s ResearchInstitute (Columbus) for support. This work was supported by two grants fromNIH (AI-42715 and AI-44974) and a grant from the American Foundation forAIDS Research (AMFAR) to K.S.

RECEIVED 9 JUNE; ACCEPTED 27 NOVEMBER 2000

1. Robey, W.G. et al. Characterization of envelope and core structural gene productsof HTLV-III with sera from AIDS patients. Science 228, 593–595. (1985).

2. Flamand, L. et al. Activation of CD8+ T lymphocytes through the T cell receptorturns on CD4 gene expression: implications for HIV pathogenesis. Proc. Natl. Acad.Sci. USA 95, 3111–3116. (1998).

3. Kitchen, S.G., Korin, Y.D., Roth, M.D., Landay, A. & Zack, J.A. Costimulation ofnaive CD8(+) lymphocytes induces CD4 expression and allows human immunodefi-ciency virus type 1 infection. J. Virol. 72, 9054–9060. (1998).

4. Saha, K., Zhang, J. & Zerhouni, B. Evidence of productively infected CD8+ T cells inpatients with AIDS: implications for HIV-1 pathogenesis (manuscript submitted).

5. Saha, K., Sova, P., Chao, W., Chess, L. & Volsky, D.J. Generation of CD4+ and CD8+

T-cell clones from PBLs of HIV-1 infected subjects using herpesvirus saimiri. Nat.Med. 2, 1272–1275 (1996).

6. Saha, K., Bentsman, G., Chess, L. & Volsky, D.J. Endogenous production of β-chemokines by CD4+, but not CD8+, T-cell clones correlates with the clinical state ofhuman immunodeficiency virus type 1 (HIV-1)-Infected individuals and may be re-sponsible for blocking infection with non-syncytium-inducing HIV-1 in vitro. J. Virol.72, 876–881 (1998).

7. LaBranche, C. et al. Determinants of CD4 independence for a human immunodefi-ciency virus type 1 variant map outside regions required for coreceptor specificity. J.Virol. 73, 10310–10319. (1999).

8. Reeves, J.D. et al. Primary human immunodeficiency virus type 2 (HIV-2) isolates in-fect CD4-negative cells via CCR5 and CXCR4: comparison with HIV-1 and simianimmunodeficiency virus and relevance to cell tropism in vivo. J. Virol. 73,7795–7804. (1999).

9. Dumonceaux, J. et al. Spontaneous mutations in the env gene of the human im-munodeficiency virus type 1 NDK isolate are associated with a CD4-independententry phenotype. J. Virol. 72, 512–519. (1998).

10. Kolchinksy, P. et al. Adaptation of a CCR5-using, primary human immunodeficiencyvirus type 1 isolate for CD4-independent replication. J. Virol. 73, 8120–8126.(1999).

11. Hung, C.S., Vander Heyden, N. & Ratner, L. Analysis of the critical domain in the V3loop of human immunodeficiency virus type 1 gp120 involved in CCR5 utilization.J. Virol. 73, 8216-8226 (1999).

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com

72 NATURE MEDICINE • VOLUME 7 • NUMBER 1 • JANUARY 2001

ARTICLES

12. Wei, X. et al. Viral dynamics in human immunodeficiency virus type 1 infection.Nature 373, 117–122 (1995).

13. Perelson, A.S., Neumann, A.U., Markowitz, M., Leonard, J.M. & Ho, D.D. HIV-1 dy-namics in vivo: virion clearance rate, infected cell life-span, and viral generationtime. Science 271, 1582–1586 (1996).

14. Hoffman, T.L., Stephens, E.B., Narayan, O. & Doms, R.W. HIV type I envelope de-terminants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors. Proc. Natl.Acad. Sci. USA 95, 11360–11365 (1998).

15. McMichael, A.J. & Phillips, R.E. Escape of human immunodeficiency virus from im-mune control. Annu. Rev. Immunol. 15, 271–296 (1997).

16. Saha, K., McKinely, G. & Volsky, D.J . Improvement of herpesvirus saimiri T cell im-mortalization procedure to generate multiple CD4+ cells clones from peripheralblood lymphocytes of AIDS patients. J. Immunol. Methods 206, 21–23 (1997).

17. Meinl, E., Hohlfeld, R., Wekerle, H. & Fleckstein, B. Immortalization of human Tcells by herpesvirus saimiri. Immunol. Today 16, 55–58 (1995).

18. Moriuchi, H., Moriuchi, M., Combadiere, C., Murphy P.M. & Fauci, A.S. CD8+ T-cell–derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β,suppress HIV-1 replication in monocyte/macrophage. Proc. Natl. Acad. Sci. USA 93,15341-15345 (1996).

19. Hwang, S.S., Boyle, T.J., Lyerly, H.K. & Cullen, B.R. Identification of the envelopeV3 loop as the primary determinant of cell tropism in HIV-1. Science 253, 71–74(1991).

20. Cao, J. et al. Replication and neutralization of human immunodeficiency virus type1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J. Virol.71, 9808–9812. (1997).

21. Koito, A., Harrowe, G. & Levy, J.A. Functional role of the V1/V2 region ofhuman immunodeficiency virus type 1 envelope glycoprotein gp120 in infec-tion of primary macrophages and soluble CD4 neutralization. J. Virol. 68,

2253–2259. (1994).22. Ross, T.M. & Cullen, B.R. The ability of HIV type 1 to use CCR-3 as a coreceptor is

controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropicV3 loop. Proc. Natl. Acad. Sci. USA 95, 7682–7686. (1998).

23. Westervelt, P. et al. Macrophage tropism determinants of human immunodefi-ciency virus type 1 in vivo. J. Virol. 66, 2577–2582 (1992).

24. Kwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complexwith the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659(1998).

25. Rizzuto, C. et al. A conserved HIV gp120 glycoprotein structure involved inchemokine receptor binding. Science 280, 1949–1953 (1998).

26. Shankarappa, R. et al. Consistent viral evolutionary changes associated with theprogression of human immunodeficiency virus type 1 infection J. Virol. 73,10489–10502 (1999).

27. Kaneko, H. et al. Human immunodeficiency virus type 2 envelope glycoproteinbinds to CD8 as well as to CD4 molecules on human T cells J. Virol. 71, 8918–8922(1997).

28. Saha, K., Ware, R., Yellin, M.J., Chess L. & Lowy, I. Herpesvirus saimiri-transformedhuman CD4+ T cells can provide polyclonal B cell help via the CD40L as well as theTNF-α pathway and through release of lymphokines. J. Immunol. 157, 3876–3885(1996).

29. Saha, K., Volsky, D.J. & Matczak, E. Resistance against Syncytium-Inducing HumanImmunodeficiency Virus Type 1 (HIV-1) in Selected CD4+ T Cells from an HIV-1-in-fected Nonprogressor: Evidence of a Novel pathway of Resistance Mediated by aSoluble Factor (s) That Acts after Virus Entry. J Virol. 73, 7891–7898 (1999).

30. Douglas, N.W. et al. An efficient method for the rescue and analysis of functionalHIV-1 env genes: evidence for recombination in the vicinity of the tat/rev splice site.AIDS 10, 39–46 (1996).

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://m

edic

ine.

nat

ure

.co

m© 2001 Nature Publishing Group http://medicine.nature.com