Novel Activities of Cyclophilin A and
Cyclosporin A during HIV-1 Infection of
Primary Lymphocytes and Macrophages
This information is current as
of June 18, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2006; 177:443-449; ;
doi: 10.4049/jimmunol.177.1.443
http://www.jimmunol.org/content/177/1/443
This article cites 34 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/177/1/443.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
Manisha Saini and Mary Jane Potash
The Journal of Immunology
Novel Activities of Cyclophilin A and Cyclosporin A during
HIV-1 Infection of Primary Lymphocytes and Macrophages1
Manisha Saini and Mary Jane Potash2
uch of our extensive knowledge of HIV type 1 (HIV1)3 biology and regulation has been obtained during
virus infection or expression in transformed cells in
culture. In many respects, primary macrophages provide a very
different cellular environment for HIV-1 replication, and as a result, specific aspects of the virus– host cell interaction are different.
For example, expression of CD4 and CXCR4 is sufficient to mediate virus entry and fusion and productive infection of many
transformed human cells (1); however, laboratory-adapted X4
HIV-1 does not productively infect primary CD4-CXCR4-bearing
human macrophages, and the restriction has been mapped, in part,
to postentry events in virus replication (2, 3). Despite the use of
CCR5 as a coreceptor on both lymphocytes and macrophages, R5
HIV-1 infection of lymphocytes can be blocked by the native ligands of CCR5 but infection of macrophages proceeds unimpeded
(4 – 6). Viral transcription also has a cell lineage-specific component, the C/EBP binding sites in the HIV-1 long terminal repeat are
essential for replication in primary macrophages but dispensable
for replication in primary lymphocytes (7). At the final stage of the
viral life cycle, macrophages display their most characteristic trait;
they direct HIV-1 assembly to intracellular membranes, so that the
viruses bud predominantly into internal membrane-bound vesicles,
rather than the plasma membrane as in lymphocytes (8). This distinction determines the cellular proteins that are incorporated into
the particle. Thus, the virions produced by the two major targets of
HIV-1 infection, lymphocytes and macrophages, differ in compo-
M
sition. In principle, this difference can influence infectivity, virulence, and sensitivity to antiviral intervention.
Cyclophilin A (CypA) is a peptidyl-prolyl isomerase of cellular
origin that is specifically incorporated into HIV-1 virions through
a well-defined interaction with Gag in capsids (9, 10). Treatment
of transformed cells with drugs that interfere with this interaction,
notably the immunosuppressive agent, cyclosporin A (CsA), reduces the incorporation of CypA into virions and inhibits HIV-1
infection (10 –13). Because of the differences between HIV-1 assembly in macrophages and assembly in other cell types, we investigated the incorporation of CypA in virions from infected primary human macrophages, compared with infected PBLs.
Using the specific requirements for assembly of replication
competent HIV-1 in transformed cells in culture as a basis, we
analyzed virion composition and infectivity as perturbed by mutation or pharmacological intervention with reference to the
progress of the viral life cycle in primary cells. Contrary to expectation, our studies demonstrate that the major distinction among
host cell types in these elements of HIV-1 infection lies between
transformed cells on the one hand and both primary lymphocytes
and macrophages on the other hand. We report that CypA–Gag
interactions, CsA sensitivity, and the biology of mutations that
disrupt these effects are different in primary cells than was reported
previously in various transformed human cell lines. Our studies
suggest caution in interpreting that activity of HIV-1 inhibitors
solely by test of their effects on virus replication in transformed
host cells.
Molecular Virology Division, St. Luke’s-Roosevelt Hospital Center, Columbia University Medical Center, New York, NY 10019
Materials and Methods
Received for publication December 13, 2005. Accepted for publication April 7, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Public Health Service Grants NS39191 and NS43110.
2
Address correspondence and reprint requests to Dr. Mary Jane Potash, Molecular
Virology Division, St. Luke’s-Roosevelt Hospital Center, 432 West 58th Street, New
York, NY 10019. E-mail address: [email protected]
3
Abbreviations used in this paper: HIV-1, HIV type 1; CypA, cyclophilin A; CsA,
cyclosporin A; MDM, monocyte-derived macrophage.
Copyright © 2006 by The American Association of Immunologists, Inc.
Cells
Human PBLs and monocytes were obtained by centrifugal elutriation from
blood collected from ⬎20 healthy individuals under an exemption from
Institutional Review Board review. CEM and 293T cells were obtained
from the AIDS Research Reagent Repository (Rockville, MD). PBLs were
stimulated with 5 ␮g/ml PHA (Sigma-Aldrich) in RPMI 1640 medium
containing 10% FBS (Omega Scientific) and 1 ng/ml IL-2 (R&D Systems)
for 48 h and then cultured without PHA. Monocytes were induced to differentiate to macrophages by culture in 10% human serum (Cambrex) and
10% giant cell tumor-conditioned medium (BioVeris) in DMEM for 5 days
and thereafter were cultured in DMEM containing 10% FBS.
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when
CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA),
HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host
proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human
macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity,
and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple
donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that
the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of
virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the
identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed
cells may not recapitulate infection of the native targets of HIV-1. The Journal of Immunology, 2006, 177: 443– 449.
444
Viruses
Infection
PBLs or monocyte-derived macrophage (MDMs) were infected with various HIV-1 at 0.1 pg p24 per cell for 1–2 h at 37°C, washed, and then
cultured in the absence or presence of 2.5 ␮M CsA (Sigma-Aldrich or
Calbiochem). Cells and cell supernatants were harvested at the times indicated for evaluation of p24 and CypA content, HIV-1 DNA or RNA, or
infectious HIV-1.
RT and PCR
Cells were collected at the indicated time points, and genomic DNA was
prepared using DNAzol (Invitrogen Life Technologies). DNA content was
standardized by amplification of the single-copy cellular ␤-globin gene
using primers as described (3). To detect HIV-1 DNA, a 115-bp region of
gag was amplified using primers SK38 (5⬘-ATAATCCACCTATCCCAG
TAGGAGAAAT-3⬘) and SK39 (5⬘-TTTGGTCCTTGTCTTATGTCCA
GAATGC-3⬘), electrophoresised, blotted, and probed using 32P-labeled
SK19 (5⬘-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCC
TAC-3⬘) (14) as described (3). Alternatively, total RNA was isolated using
TRIzol (Invitrogen Life Technologies) by following the manufacturer’s
instructions. Four micrograms of the RNA was used to prepare cDNA
(Superscript First Strand synthesis kit for RT-PCR; Invitrogen Life Technologies) from the doubly spliced vif message of HIV using the primer
VFSP (5⬘-AACCAGTCCTTAGCTTTCCTTGAAATATAC-3⬘). Two microliters of the cDNA was used to amplify a 374-bp region of the vif
message using the forward primer aligning in the long terminal repeat
region, NL616 (5⬘-GTGTGGAAAATCTCTAGCAGTGGCGC-3⬘), and
the reverse primer VFSP, and probed with VF5071 (5⬘-GGCAAGTA
GACAGGATGAGGA-3⬘).
by loading 10 –50 ng of p24; the bands were transferred to a polyvinylidene
difluoride (Millipore) membrane. Bands were visualized by Western blotting using standard procedure and staining with anti-CypA Ab (Affinity
BioReagents), HRP-conjugated anti-human Ab, and the detection by
chemiluminescence using p-coumaric acid (4-hydroxycinnamic acid; Sigma-Aldrich) and luminol (5-amino-2,3-dihydro-1,4-pthalazinedione; Sigma-Aldrich). The membrane was stripped with 0.2 M sodium hydroxide
for 5 min, washed with PBS, and used to detect p24 protein using a mouse
monoclonal anti-HIV-1 CA (catalog no. 183-H12-5C) provided by Dr. B.
Chesebro (Rocky Mountain National Laboratories, Hamilton, MT) and Dr.
K. Wehry (Rocky Mountain National Laboratories, Hamilton, MT) through
the AIDS Research Reagent Repository.
MAGI assay
The MAGI assay was conducted as described by Kimpton and Emerman
(15) using virions isolated from infected cells and standardized by p24
content. MAGI cells were provided by Dr. M. Emerman (University of
Washington, Seattle, WA). For assay of infectivity of R5 viruses, MAGICCR5 cells, provided by Dr. J. Overbaugh (University of Washington,
Seattle, WA), were used. Both cells lines were distributed by the AIDS
Research Reagent Repository.
Results
CypA incorporation into virions produced by HIV-1-infected
primary macrophages and lymphocytes
We investigated the CypA incorporation into virions isolated from
NL4-3-infected PBLs or ADA-infected MDMs by Western blot. In
this study, all samples for Western blot are first standardized by
p24 content by ELISA to facilitate a direct comparison of samples.
MDMs obtained from three donors and PBLs from two of the same
donors were infected in culture, and virions isolated at different
times after infection were compared for CypA content (Fig. 1).
The CypA incorporation into virions was variable, depending on
the cell donor and time after infection, and virions from MDM
contained less CypA (Fig. 1A) than those from PBLs from the
same donors (Fig. 1B). CypA content appeared to decrease over
the course of infection of MDM but increase over the course of
infection of PBLs (see Fig. 2B). This variable incorporation suggested that the association of CypA with Gag is not tightly regulated in primary cells and may differ from that in transformed cells
(16).
We then used pharmacological intervention with CsA to inhibit
the Gag–CypA association. Cell viability was tested during culture
of both uninfected and HIV-1-infected PBLs and MDMs in the
absence and presence of CsA. CsA was not toxic under the conditions used in this study in which it is only added to cells after
Analysis of virion protein composition
Cell supernatants containing HIV-1 were centrifuged at 300 ⫻ g, and the
virions in the cell-free medium was sedimented at 19,500 rpm in a rotor
(model J20.1; Beckman Coulter) for 2 h. The pellet was either analyzed
directly or used for a second round of purification through sucrose gradient.
A discontinuous sucrose gradient of 600 ␮l of 30%, 200 ␮l of 20% containing 0.5% Triton X-100, and 200 ␮l of 10% was layered on top of the
concentrated virus sample and centrifuged at 14,000 rpm in a centrifuge
(model 5402; Eppendorf) at 4°C for 3 h. The virion pellets were resuspended in PBS, titered for p24 content, and then run on 15% SDS-PAGE
FIGURE 1. CypA content in virions produced by HIV-1-infected
MDMs or PBLs. Virions were isolated from culture supernatants of ADAinfected MDM from three different donors at two time points or of NL43-infected PBLs from two different donors on day 7 after infection and the
capsid Ag p24 content was determined by ELISA. From 20 to 100 ng p24
of MDM-derived virions (A), or 20 – 60 ng p24 of PBL-derived virions (B)
were loaded per lane as indicated and subjected to electrophoresis and
Western blot, staining for CyA.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
NL4-3 was propagated in CEM cells and isolated during acute infection.
Subtype A/E CM235 was obtained from Dr. N. Michael (Walter Reed
Army Institute of Research, Rockville, MD) through the AIDS Research
Reagent Repository and was propagated in PBLs and isolated during acute
infection. pA224E, a plasmid encoding CypA-resistant mutant of NL4-3
was a gift from Dr. J. Luban (Columbia University Medical Center, New
York, NY). pNLHXADA-GP was a gift from Dr. L. Ratner (Washington
University, St. Louis, MO). The plasmids were propagated in DH5␣ cells
and transfected into 293T cells using LipofectAMINE (Invitrogen Life
Technologies) according to the manufacturer’s instructions, and infectious
virions were isolated from supernatant 48 and 72 h after transfection. ADA
was a gift from Dr. H.E. Gendelman (University of Nebraska Medical
Center, Omaha, NE) and was propagated exclusively in primary macrophages and isolated from their supernatants. A mutant of NLHXADA-GP
carrying the A224E mutation was prepared by PCR, carrying out the single
point mutation of C to A so as to change the amino acid from alanine to
glutamic acid at position 224 of the gag protein, and the mutation was
confirmed by sequencing. The mutation was conducted by amplifying the
4.3-kb fragment of the plasmid between SphI and EcoRI, with the mutation
in the forward primer MUT5 (5⬘-TCCAGTGCATGCAGGGCCTATT
GAACCAGGCCAGATGAG-3⬘). The reverse primer NL5727 (5⬘-TTGT
TGCAGAATTCTTATGGCTTCCAC-3⬘) contained the EcoRI site. The
restriction sites are underlined, and the point mutation is mentioned in bold
letters in the primers. The PCR was conducted using AccuPrime Pfx DNA
polymerase (Invitrogen Life Technologies) by denaturing the template at
95°C for 2 min initially and 30 s in the subsequent cycles, annealing at
55°C for 30 s and polymerization at 68°C for 4.5 min for 30 cycles, followed by a 7-min extension at 68°C. The PCR product was digested with
SphI and EcoRI and ligated with similarly digested and purified NLHXADA-GP. The new plasmid carrying the point mutation was called
GP-M. The mutation was confirmed by sequencing. The p24 content of all
virus stocks was determined by ELISA using the HIV-1 p24 Ag assay
(Beckman Coulter), according to the manufacturer’s instructions. For analysis of infection by HIV-1 DNA levels by PCR, the virus inoculum was
treated with DNase I as described before infection (3).
PHASES OF CsA SENSITIVITY OF HIV-1 REPLICATION
The Journal of Immunology
445
Table II. CsA inhibits HIV-1 infection of primary macrophagesa
Donor
Untreated
CsA
D
E
F
G
3.7 ⫻ 10
2.1 ⫻ 106
1.4 ⫻ 106
7.8 ⫻ 105
2.2 ⫻ 10
2.7 ⫻ 104
7.6 ⫻ 103
3.1 ⫻ 103
6
Fold Inhibition
4
171
77
185
252
a
MDM from four different donors were infected by ADA and then cultured in the
absence or presence of CsA. Core antigen p24 as determined by ELISA of cell supernatant harvested 7 days after infection is shown.
culture in mitogen or differentiation factors (not shown). We exposed MDMs to ADA or to NLHXADA-GP, a molecular clone
that carries the ADA V3 region on the background of NL4 –3
Gag-Pol (17) for 2 h, washed the cells, and then cultured them in
the presence of CsA; this experimental format was designed to
focus on postbinding events in virus replication. During infection
in culture, MDMs retain most of the capsid protein produced inside
cells, so we tested the levels of both intracellular and extracellular
p24; (Table I). The replication of both viruses was inhibited up to
5000-fold by CsA. We then tested the generality of this observation by using MDMs from four different donors for infection by
ADA, as modulated by culture in CsA (Table II). In each case,
CsA was a potent inhibitor of infection. Using cells from ⬎10
different donors and multiple HIV-1 stocks, we have found that
CsA consistently inhibits p24 expression by primary MDMsin culture (data not shown). The extent of inhibition of viral protein
production by infected MDMs was significantly greater than that
Table I. CsA stably inhibits infection of MDM using two different strains of HIV-1a
Day 7
Day 9
Day 11
Treatment
ex
int
ex
int
ex
int
ADA
ADA ⫹ CsA
GP
GP ⫹ CsA
2.4 ⫻ 106
1.2 ⫻ 104
6.7 ⫻ 104
3.2 ⫻ 103
4.6 ⫻ 107
7.3 ⫻ 104
2.2 ⫻ 107
5.0 ⫻ 104
1.1 ⫻ 106
1.8 ⫻ 103
7.2 ⫻ 104
240
4.7 ⫻ 107
1.7 ⫻ 104
1.1 ⫻ 107
2.4 ⫻ 104
4.5 ⫻ 106
2.0 ⫻ 104
3.8 ⫻ 105
2.2 ⫻ 103
8.4 ⫻ 107
2.1 ⫻ 105
6.3 ⫻ 107
9.6 ⫻ 104
a
MDM were infected with ADA or NLHXADA-GP and then cultured in the absence or presence of CsA. The levels of p24
obtained from ELISA of cells and supernatants harvested at the indicated times after infection are shown, designated intracellular
(int) or extracellular (ex), respectively.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. CypA content in virions produced by HIV-1-infected
MDMs or PBLs after culture in CsA. A, NL4 –3-infected CEM cells, NL43-infected PBLs, and ADA-infected MDMs were cultured in the absence or
presence of 2.5 ␮M CsA. Virions from infected cells were isolated and
analyzed by Western blot as described in the legend to Fig. 1. B, Virions
were isolated from NL4-3-infected PBLs cultured in the absence and presence of CsA at the times indicated, and a portion were subjected to sedimentation through a discontinuous sucrose density gradient before electrophoresis and Western blotting. Blots were stained first with an anti-CypA
antiserum, then stripped and stained with an anti-CA mAb.
previously reported for infected transformed cell lines, which was
on the order of 3- to 10-fold (12) or that previously reported for
infected PBLs using the CsA analog, SDZ MIN 811, that inhibited
4- to 20-fold (18). This consistent inhibition of HIV-1 replication
by CsA was not reflected in the variable levels of CypA found in
virions from infected primary cells (Fig. 1), raising the possibility
that CypA incorporation is not the only event in virus infection
sensitive to CsA. To directly link these physiological findings to
virion composition, the CypA content of virions isolated from infected PBLs or MDMs that had been cultured in the presence or
absence of CsA was determined. Cells were obtained from two
different donors. Virions from infected transformed CEM cells
were run in parallel, all samples were standardized by p24 content
(Fig. 2A). Consistent with previous studies, culture of infected
CEM cells in CsA significantly inhibited CypA incorporation into
virions. In contrast, there was little to no inhibition of CypA incorporation into virions derived from primary cells. Despite standardization by p24 content, virions from highly viable MDMs
seemed to show enhanced incorporation of CypA after CsA treatment. To evaluate a more highly purified population of virions for
CypA content, we repeated this experiment using PBLs from another donor and subjected virions to sucrose density gradient sedimentation (Fig. 2B). Based on p24 content, sucrose gradient-purified virions had more CypA than the starting virion population.
However, CsA treatment of virion-producing cells increased rather
than decreased CypA content, and gradient purification further increased the relative CypA content of virions. These studies from a
total of five different macrophage and five different PBL donors
indicate the CypA incorporation into virions by primary lymphocytes and macrophages is regulated differently than those of transformed cells
Many previous studies have shown that the CypA–Gag association, and thus CypA virion incorporation, is blocked by CsA and
that this reduction in the CypA content of virions renders them less
infectious (10, 12, 13). Our finding in primary cells that CsA is a
potent inhibitor of HIV-1 infection but does not decrease CypA
content of virions is inconsistent with this observation. To directly
test the proposal, we isolated virions from infected cells cultured in
the presence or absence of CsA and tested their infectivity by
446
PHASES OF CsA SENSITIVITY OF HIV-1 REPLICATION
MAGI assay of single cycle infection or by assay of p24 production by infected PBL (Fig. 3). When standardized by p24 levels,
several different HIV-1 produced in the presence of CsA were
equally as infectious as those produced without inhibitor. CsA derived HIV-1 of both subtype B and A/E or R5 and X4 tropism were
found to be infectious. Similar results have been obtained using
three different cell donors and virus stocks. To better understand
this apparent CypA independence but CsA sensitivity of HIV-1
infection of primary cells, we used an HIV-1 mutated in the CypA
binding region.
Sites of restriction by CsA treatment of HIV-1-infected primary
cells as a function of Gag sequence
FIGURE 4. Efficiency of infection of PBLs or MDMs by wild-type or
A224E mutated HIV-1 as perturbed by culture in CsA. PBLs were infected
with NL4 –3 or A224E (A), and MDMs were infected by NLHXADA-GP
or NLHXADA-GP/A224E (B) and then cultured in the absence or presence
of CsA. Cells or supernatants were harvested at the indicated times after
infection, supernatants were replaced by fresh medium, and the extracellular p24 content from PBLs or the intracellular p24 content of MDMs was
determined by Elisa.
to A224E replication in PBL. The results of A224E infection reveal a CsA-sensitive postentry phase in HIV-1 replication in primary cells absent from transformed T cell lines.
To investigate the basis of this inhibition by CsA, we tested viral
DNA synthesis over time after infection by PCR, amplifying a
FIGURE 3. Infectivity of virions isolated from HIV-1-infected, CypAtreated PBLs. Virions produced by HIV-1-infected PBLs cultured in the
absence or presence of CsA were standardized by p24 content and tested
for infectivity by MAGI assay (A) or by infection of PBLs and measurement of production of core Ag p24 (B).
FIGURE 5. Viral DNA synthesis by HIV-1-infected PBLs or MDMs as
perturbed by culture in CsA. PBLs were infected by DNase-treated NL4-3
or A224E (A) or MDMs were infected by DNase-treated ADA (B) and then
cultured in the absence or presence of CsA. Cells were harvested at the
indicated times after infection, DNA was isolated and subjected to PCR
amplification of regions in the cellular ␤-globin gene or the HIV-1 gag
gene. The ␤-globin amplicon was visualized by ethidium bromide staining,
and the gag amplicon was visualized by hybridization with radiolabeled
probe.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
To clarify the link between CsA inhibition and CypA in HIV-1infected primary cells, we used A224E, an NL4 –3 variant that
emerged after propagation in the presence of CsA but that still
encapsidates CypA into virions. This point mutation replaces an
uncharged residue with a charged residue in a CypA binding region of Gag and has been shown to render HIV-1 infection in
transformed Jurkat cells resistant to CsA and infection in transformed CEM or MAGI cells dependent on CsA (12, 13, 16). We
first tested the replication of A224E, compared with NL4-3 by
culture of infected PBLs in the absence and presence of CsA and
assay of extracellular p24 over time (Fig. 4A). Surprisingly, A224E
replication in PBLs was as sensitive to CsA inhibition as that of
wild-type NL4-3. Unlike CEM or MAGI cells, PBL infection by
A224E was neither dependent nor enhanced by CsA; unlike Jurkat,
PBL infection was not resistant to CsA. Similar results were obtained using PBLs from three other donors (data not shown). Analogous studies performed in MDMs using an A224E mutation in
NLHXADA-GP yielded similar findings (Fig. 4B) in MDMs from
some, but not all, donors, so further studies here will be restricted
The Journal of Immunology
FIGURE 6. Viral RNA synthesis by HIV-1-infected PBLs as perturbed
by culture in CsA. PBLs were infected by NL4-3 or A224E and then
cultured in the absence or presence of CsA. Total cellular RNA was isolated at the indicated times, and a fixed amount of RNA was subjected to
RT-PCR, amplifying a region in the singly spliced viral vif mRNA. The vif
amplicon was visualized by hybridization with a radiolabeled probe.
PBLs (Fig. 7). We found that CsA inhibited A224E and NL4-3 p24
accumulation in both compartments, ruling out a block to virion
export, but consistent with a block at or near the level of translation. We tested the infectivity of A224E virions produced in the
absence and presence of CsA, with NL4-3 run in parallel for reference, standardizing infectious dose by p24 content (Fig. 8). Although CsA is a potent inhibitor of HIV-1 infection in PBLs and
greatly reduces the production of progeny virions, it had little effect upon the infectivity of the progeny virus produced. It should
be noted that CsA was shown to enhance A224E replication in
MAGI cells (12, 13, 16), as confirmed in the assay performed in
this study. Taken as a whole, the findings reported in this study
indicate that CsA inhibits an early phase in HIV-1 replication in
primary cells before reverse transcription, and that this sensitivity
is controlled by Gag. Mutants in Gag that evade the first block are
subject to a second phase of inhibition by CsA at the level of viral
protein production.
Discussion
Our results demonstrate that HIV-1 replication in primary cells is
sensitive to the multifunctional inhibitor CsA during two processes: before reverse transcription and at protein production, the phase
affected depends on sequences within Gag. Inhibition of virus production appears to be independent of CypA, and CypA itself is
variably associated with virions. These findings are quite different
from earlier studies of HIV-1 infectivity regarding a requirement
for incorporation of CypA into virions for its function (19), but are,
in part, consistent with recent studies using transformed cells that
place the major function for CypA at early but not late stages of
HIV-1 replication (20, 21). Our work extends these studies by
suggesting that the interaction of Gag with cellular proteins in
FIGURE 7. Efficiency of viral protein export from PBLs infected by wild-type or A224E-mutated HIV-1 as perturbed by culture in CsA. PBLs were
infected by NL4-3 (A and C) or A224E (B and D) and then cultured in triplicate in the absence or presence of CsA. Cells and supernatants were harvested
at the times indicated and extracellular (A and B) or intracellular p24 (C and D) were measured by ELISA. Means ⫾ SDs are shown.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
region in gag from NL4-3- and A224E-infected PBLs or ADAinfected MDMs cultured in the absence or presence of CsA (Fig.
5). Consistent with inhibition of synthesis of viral protein, NL4-3
DNA synthesis in PBLs and ADA DNA synthesis in MDMs was
inhibited by CsA treatment. In contrast, A224E DNA synthesis
was not affected by CsA treatment. To probe the next phase of the
A224E life cycle in PBL, we tested viral RNA production by RTPCR, amplifying the singly spliced vif mRNA in PBL infected and
cultured under the same conditions (Fig. 6). Once again, there was
a clear distinction between NL4-3 infection and A224E infection;
NL4 –3 RNA production was almost completely inhibited by CsA
treatment, but A224E transcription was less sensitive to CsA, declining only at the later time points relative to untreated infected
cells. These findings narrow the possible activities of CsA in
A224E-infected cells either to inhibition of viral protein production or to impairment of the infectivity of progeny virus. To distinguish a block in virus assembly or export from a block in virus
protein production. we repeated the experiment shown in Fig. 4A
and measured both extracellular and intracellular p24 in infected
447
448
many transformed cell lines does not predict these interactions in
primary cells and opens the possibility that antiviral interventions
proceed in the native target cells of HIV-1 differently than in transformed cells.
HIV-1 assembly and virion content are different in infected macrophages than other cell types. Virions assemble in late endosomes
in macrophages and contain cellular proteins characteristic of this
compartment, LAMP-1, CD81, and CD82 rather than cell surface
markers like CD11a or CD14 (22, 23); virions exit the cell through
exocytosis. In contrast, in primary and transformed T cells, virion
components assemble at and bud directly from the plasma membrane. We investigated whether CypA incorporation may also distinguish HIV-1 virions produced by infected macrophages from
those produced by lymphocytes. Contrary to expectation, both primary lymphocytes and macrophages produced virions with low
and variable levels of CypA; a virion composition different from
that observed from infection of transformed cells in culture (10 –
13). Our observation that PBL- or MDM-derived infectious HIV-1
virions sometimes contain little to no CypA is supported by recent
studies showing that transformed cells lacking CypA can produce
infectious HIV-1 virions (21).
These results question the involvement of CypA in HIV-1 infection of primary cells, however they pertain only to late stages of
virus assembly. CsA is a multifunctional immunosuppressive
agent (24) that was shown to disrupt the association between Gag
and CypA and has frequently been used to probe this protein–
protein interaction. We used untreated virus for infection and then
cultured infected PBLs or MDMs in the absence or presence of
CsA to investigate the sensitivity of early events in HIV-1 replication. This approach revealed a profound block at or before reverse transcription of wild-type HIV-1 in both lymphocytes and
macrophages, consistent with the block described in transformed
cells (19). Although the extent of the defect is not precisely quantified, CsA appears to exert a greater effect upon infection of primary cells than the 3- to 10-fold reduction observed in most transformed cells (13, 19). In the absence of efficient viral DNA
synthesis, later synthesis of viral RNA and protein also was greatly
reduced in primary cells. However, CsA treatment of infected primary cells not only did not reduce the CypA content of virions, but
also, in some cases, increased CypA content of virions, dissociating the inhibitory activity of CsA from the CA-driven incorporation of CypA into virions.
Although CsA treatment greatly reduces the quantity of virions
produced by PBLs or MDMs by inhibiting events before wild-type
viral DNA synthesis, the inhibition is not absolute, and the small
quantity of progeny virus that is produced has unimpaired infectivity. This is a clear demonstration that the major effect of CsA on
wild-type HIV-1 is felt in the target cell and not in the virusproducing cell. Results reaching the same conclusion by different
means have been obtained in some transformed cells (20, 21). Our
studies do not permit us to unambiguously identify the CsA-sensitive step early in infection. Virion uncoating has been proposed
as a CypA-dependent, CsA-sensitive step in HIV-1 replication in
transformed cells by some investigators (19) but has been ruled out
by others (25, 26). Several different cell and in vitro systems were
used for these studies. Because viral mutants in the CypA binding
region of Gag have been shown to behave differently in different
transformed cells, this distinction between experimental approaches can be biologically significant (13, 16, 21). An independent indication of fundamental differences among model systems
is the observation that a viral vector incorporating Gag from a
macrophage-tropic HIV-1 has reduced sensitivity to CsA in certain
human and simian cell lines (27), while the present study shows
that two macrophage-tropic HIV-1 are profoundly inhibited by
CsA during replication in primary human macrophages.
Our studies strongly indicate that there are intrinsic differences
between HIV-1 virion infectivity and production in primary lymphocytes and those in transformed cells; this is most clearly seen
in infection by the Gag mutant A224E. A224E infection of PBLs
can be inhibited by CsA at the stage of production of viral protein.
Viral DNA and RNA synthesis are unaffected by treatment of infected cells with CsA. This behavior is strikingly different from
that described during infection or transfection of transformed cells,
in which A224E is resistant to CsA (12, 13, 16) and also is different from PBL infection by wild-type NL4-3 in which an early
event in virus replication is sensitive to CsA. These findings indicate that factors that distinguish primary from transformed lymphocytes govern a critical interaction with HIV-1 Gag during viral
protein production. In addition, the demonstration of CsA sensitivity of A224E replication, deemed CsA resistant (12, 13, 16),
indicates that the activity of HIV-1 inhibitors is best evaluated in
primary host cells of the virus.
Indeed, natural experiments are consistent with the ability of
CsA to block HIV-1 infection in its human host. Because of the
widespread use of CsA as an immunosuppressive agent during
organ transplant, data have been collected on the course of HIV-1
infection in patients under CsA treatment. HIV-1-infected transplant recipients receiving CsA were found to have a significantly
reduced risk of progression to AIDS, compared with similar patients not treated with CsA (28). It is possible that this protective
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. Infectivity of virions isolated from NL4 –3 or A224E-infected,
CsA-treated PBLs. PBLs were infected by A224E and then cultured in the
absence or presence of CsA. A, Progeny virions were sedimented from culture
supernatants and then used to infect MAGI cells, and the MAGI assay itself
was conducted in the absence or presence of CsA, because CsA was shown to
enhance A224E replication in these cells (12). B, Alternatively, infected PBLs
were cultured in the absence or presence of CsA, and their progeny virus was
used to infect another PBL culture in the absence of CsA. At the times indicated, culture medium was harvested and replaced, and infection was monitored by measurement of extracellular p24 by ELISA.
PHASES OF CsA SENSITIVITY OF HIV-1 REPLICATION
The Journal of Immunology
Acknowledgments
We thank W. Chao for expert advice, Dr. D. J. Volsky for invaluable
discussions, and I. Totillo for manuscript preparation. We also thank the
following individuals for gifts of reagents: Dr. J. Luban for pA224E, Dr. L.
Ratner for pNLHXADA-GP, Dr. H. E. Gendelman for ADA, Dr. B. Chesebro and Dr. K. Wehry for hybridoma 183-H12-56, Dr. M. Emerman for
MAGI cells, Dr. N. Michael for CM235, and Dr. J. Overbaugh for CCR5
MAGI cells. The last four reagents were distributed through the AIDS
Research Reagent Repository.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Disclosures
The authors have no financial conflict of interest.
24.
References
25.
1. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry
cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled
receptor. Science 272: 872– 877.
2. Schmidtmayerova, H., M. Alfano, G. Nuovo, and M. Bukrinsky. 1998. HIV-1
T-lymphotropic strains enter macrophages via a CD4- and CXCR4-mediated
pathway: replication is restricted at a postentry level. J. Virol. 72: 4633– 4642.
3. Chowdhury, I. H., G. Bentsman, W. Choe, M. J. Potash, and D. J. Volsky. 2002.
The macrophage response to HIV-1: intracellular control of X4 virus replication
accompanied by activation of chemokine and cytokine synthesis. J. Neuro Virol.
8: 599 – 610.
4. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy,
P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤
receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:
1955–1958.
5. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima,
C. Cayabab, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996.
HIV-1 entry into CD4⫹ cells is mediated by the chemokine receptor CC-CKR-5.
Nature 381: 667– 673.
6. Schmidtmayerova, H., B. Sherry, and M. Bukrinsky. 1996. Chemokines and HIV
replication. Nature 382: 767.
7. Henderson, A. J., and K. L. Calame. 1997. CCAAT/enhancer binding protein
(C/EBP) sites are required for HIV-1 replication in primary macrophages but not
CD4⫹ T cells. Proc. Natl. Acad. Sci. USA 94: 8714 – 8719.
8. Orenstein, J. M., M. S. Meltzer, T. Phipps, and H. E. Gendelman. 1988. Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1
and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. J. Virol. 62: 2578 –2586.
9. Martinez-Martinez, S., and J. M. Redondo. 2004. Inhibitors of the calcineurin/
NFAT pathway. Curr. Med. Chem. 11: 997–1007.
10. Franke, E. K., H. E. H. Yuan, and J. Luban. 1994. Specific incorporation of
cyclophilin A into HIV-1 virions. Nature 372: 359 –362.
11. Billich, A., F. Hammerschmid, P. Peichl, R. Wenger, G. Zenke, V. Quesniaux,
and B. Rosenwirth. 1995. Mode of action of SDZ NIM 811, a nonimmunosup-
26.
27.
28.
29.
30.
31.
32.
33.
34.
pressive cyclosporin A analog with activity against human immunodeficiency
virus (HIV) type 1: interference with HIV protein– cyclophilin A interactions.
J. Virol. 69: 2451–2461.
Aberham, C., S. Weber, and W. Phares. 1996. Spontaneous mutations in the
human immunodeficiency virus type 1 gag gene that affect viral replication in the
presence of cyclosporins. J. Virol. 70: 3536 –3544.
Braaten, D., C. Aberham, E. K. Franke, L. Yin, W. Phares, and J. Luban. 1996.
Cyclosporin A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A. J. Virol. 70:
5170 –5176.
Ou, C.-Y., S. Kwok, S. Mitchell, D. Mack, J. Sninsky, J. Krebs, P. Feorino,
D. Warfield, and G. Schocketman. 1988. DNA amplification for direct detection
of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239: 295–297.
Kimpton, J., and M. Emerman. 1992. Detection of replication-competent and
pseudotyped human immunodeficiency virus with a sensitive cell line on the basis
of activation of an integrated ␤-galactosidase gene. J. Virol. 66: 2232–2239.
Yin, L., D. Braaten, and J. Luban. 1998. Human immunodeficiency virus type 1
replication is modulated by host cyclophilin A expression levels. J. Virol. 72:
6430 – 6436.
Westervelt, P., H. E. Gendelman, and L. Ratner. 1991. Identification of a determinant within the human immunodeficiency virus 1 surface envelope glycoprotein critical for productive infection of primary monocytes. Proc. Natl. Acad. Sci.
USA 88: 3097–3101.
Mlynar, E., D. Bevec, A. Billich, B. Rosenwirth, and A. Steinkasserer. 1997. The
non-immunosuppressive cyclosporin A analogue SDZ NIM 811 inhibits cyclophilin A incorporation into virions and virus replication in human immunodeficiency virus type 1-infected primary and growth-arrested T cells. J. Gen. Virol.
78 (Pt. 4): 825– 835.
Braaten, D., E. K. Franke, and J. Luban. 1996. Cyclophilin A is required for an
early step in the life cycle of human immunodeficiency virus type 1 before the
initiation of reverse transcription. J. Virol. 70: 3551–3560.
Sokolskaja, E., D. M. Sayah, and J. Luban. 2004. Target cell cyclophilin A
modulates human immunodeficiency virus type 1 infectivity. J. Virol. 78:
12800 –12808.
Hatziioannou, T., D. Perez-Caballero, S. Cowan, and P. D. Bieniasz. 2005. Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells. J. Virol. 79: 176 –183.
Pelchen-Matthews, A., B. Kramer, and M. Marsh. 2003. Infectious HIV-1 assembles in late endosomes in primary macrophages. J. Cell Biol. 162: 443– 455.
Esser, M. T., D. R. Graham, L. V. Coren, C. M. Trubey, J. W. Bess, Jr.,
L. O. Arthur, D. E. Ott, and J. D. Lifson. 2001. Differential incorporation of
CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class
I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation. J. Virol.
75: 6173– 6182.
Gelfand, E. W., R. K. Cheung, and G. B. Mills. 1987. The cyclosporins inhibit
lymphocyte activation at more than one site. J. Immunol. 138: 1115–1120.
Wiegers, K., G. Rutter, U. Schubert, M. Grättinger, and H.-G. Kräusslich. 1999.
Cyclophilin A incorporation is not required for human immunodeficiency virus
type 1 particle maturation and does not destabilize the mature capsid. Virol. 257:
261–274.
Grattinger, M., H. Hohenberg, D. Thomas, T. Wilk, B. Muller, and
H. G. Krausslich. 1999. In vitro assembly properties of wild-type and cyclophilinbinding defective human immunodeficiency virus capsid proteins in the presence
and absence of cyclophilin A. Virol. 257: 247–260.
Kootstra, N. A., C. Münk, N. Tonnu, N. R. Landau, and I. M. Verma. 2003.
Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction
in simian cells. Proc. Natl. Acad. Sci. USA 100: 1298 –1303.
Schwarz, A., G. Offermann, F. Keller, I. Bennhold, J. L’Age-Stehr, P. H. Krause,
and M. J. Mihatsch. 1993. The effect of cyclosporin on the progression of human
immunodeficiency virus type 1 infection transmitted by transplantation: data on
four cases and review of the literature. Transplantation 55: 95–103.
Sheehy, A. M., N. C. Gaddis, J. D. Choi, and M. H. Malim. 2002. Isolation of a
human gene that inhibits HIV-1 infection and is suppressed by the viral Vif
protein. Nature 418: 646 – 650.
Simon, J. H. M., T. E. Southerling, J. C. Peterson, B. E. Meyer, and M. H. Malim.
1995. Complementation of vif-defective human immunodeficiency virus type 1
by primate, but not nonprimate, lentivirus vif genes. J. Virol. 69: 4166 – 4172.
Ribeiro, A. C., A. Maia e Silva, M. Santa-Marta, A. Pombo, J. Moniz-Pereira,
J. Goncalves, and I. Barahona. 2005. Functional analysis of Vif protein shows
less restriction of human immunodeficiency virus type 2 by APOBEC3G. J. Virol. 79: 823– 833.
Towers, G. J., T. Hatziioannou, S. Cowan, S. P. Goff, J. Luban, and
P. D. Bieniasz. 2003. Cyclophilin A modulates the sensitivity of HIV-1 to host
restriction factors. Nat. Med. 9: 1138 –1143.
Berthoux, L., S. Sebastian, E. Sokolskaja, and J. Luban. 2005. Cyclophilin A is
required for TRIM5␣-mediated resistance to HIV-1 in Old World monkey cells.
Proc. Natl. Acad. Sci. USA 102: 14849 –14853.
Sokolskaja, E., L. Berthoux, and J. Luban. 2006. Cyclophilin A and TRIM5-␣
independently regulate human immunodeficiency virus type 1 infectivity in human cells. J. Virol. 80: 2855–2862.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
effect is due to the block in virus infection of primary cells demonstrated in this study.
The present study, like many others, indicates that cell typespecific factors can greatly influence the outcome of HIV-1 infection. One such factor is APOBEC3G, which was shown to antagonize the activity of HIV-1 Vif; its expression was postulated to
underlie the difference among some target cells lines in their susceptibility to Vif-negative HIV-1 (29). Curiously, although HIV-2
Vif can complement Vif-negative HIV-1 (30), it is poorly antagonized by APOBEC3G, and cell lines expressing APOBEC3G can
be susceptible to Vif-negative HIV-2 but not to Vif-negative
HIV-1 (31). Different species-specific host restriction factors have
been proposed to interact with the CypA-binding domain of CA
during early phases of HIV-1 infection of certain nonpermissive
simian cells (21, 32, 33). The analogous human factor, TRIM 5-␣,
also was postulated to act through CypA, but recent studies demonstrate its independence from CypA in human cells (34). This
observation underscores the difficulties of extrapolating HIV-1 behavior from model systems to human lymphocytes. Our findings of
the activities of CypA and CsA during early and late stages of
HIV-1 infection of primary lymphocytes and macrophages recommend reevaluation of the interactions of viral proteins with host
proteins in the native targets of HIV-1 infection.
449