Different mutational pathways to CXCR4 coreceptor switch of CCR5-using simian-human immunodeficiency virus.
ABSTRACT We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N))-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIV(CA28NP)) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIV(CA28NL)) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIV(SF162P3N) isolate in vivo.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: We have examined the influence of the V1/V2 region of the human immunodeficiency virus type 1 (HIV-1) gp120 on certain biologic properties of the virus. We observed that on the genomic background of the T-cell-line-tropic strain, HIV-1SF2mc, both the V1 and V2 domains of the macrophage-tropic strain, HIV-1SF162mc, in addition to the required V3 domain, are necessary to attain full macrophage tropism. Furthermore, the V2 domain modulates the sensitivity of HIV-1 to soluble CD4 neutralization. Structural studies of recombinant and mutant envelope glycoproteins suggest that the function of the V1/V2 region is to interact with the V3 domain and confer on the envelope gp120 of HIV-1SF2mc a conformation more similar to that of the macrophage-tropic strain HIV-1SF162mc. The conformation of the envelope gp120 appears to be strain specific and plays an important role in determining HIV-1 tissue tropism and sensitivity to soluble CD4 neutralization.Journal of Virology 05/1994; 68(4):2253-9. · 5.08 Impact Factor
- AIDS 02/1997; 11 Suppl A:S3-16. · 6.41 Impact Factor
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ABSTRACT: The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of gp120 and gp41. gp120 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes. Previous studies identified an alpha-helical domain within gp41 composed of a trimer of two interacting peptides. The crystal structure of this complex, composed of the peptides N36 and C34, is a six-helical bundle. Three N36 helices form an interior, parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure shows striking similarity to the low-pH-induced conformation of influenza hemagglutinin and likely represents the core of fusion-active gp41. Avenues for the design/discovery of small-molecule inhibitors of HIV infection are directly suggested by this structure.Cell 05/1997; 89(2):263-73. · 31.96 Impact Factor
JOURNAL OF VIROLOGY, June 2008, p. 5653–5656
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 11
Different Mutational Pathways to CXCR4 Coreceptor Switch of
CCR5-Using Simian-Human Immunodeficiency Virus?
Siu-hong Ho,1Nataliya Trunova,1Agegnehu Gettie,1James Blanchard,2and Cecilia Cheng-Mayer1*
Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Ave., 7th Floor, New York, New York 10016,1
and Tulane National Primate Research Center, Tulane University Medical Center, 18702 Three Rivers Road,
Covington, Louisiana 704332
Received 20 January 2008/Accepted 25 March 2008
We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N
(SHIVSF162P3N)-infected macaque CA28, supporting the use of this experimental system to examine factors that
drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIVCA28NP) used both
CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIVCA28NL) used CXCR4 almost
exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4
usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent
mutational pathways for change in the coreceptor preference of the R5 SHIVSF162P3Nisolate in vivo.
In addition to the requirement for CD4, human immunode-
ficiency virus type 1 (HIV-1) requires a secondary receptor,
CCR5 or CXCR4, for entry into target cells (1). Viruses iso-
lated from newly infected individuals predominantly use CCR5
(R5 viruses), whereas CXCR4-using (X4) viruses emerge in
?50% of subtype B-infected patients with advanced disease
(7). The cause of, mechanistic basis for, and obstacles to co-
receptor switch in HIV-1 infection remain incompletely de-
fined. Factors such as immune responses directed at the enve-
lope and target cell availability are likely to influence the
probability of X4 virus evolution and selection (8).
We recently reported coreceptor switching in BR24, one of
three rhesus macaques (RM) infected with CCR5-tropic
simian-human immunodeficiency virus SF162P3N (SHIVSF162P3N)
(5), mirroring what happens during the course of HIV-1 infec-
tion. Infection of RM with SHIVSF162P3N, therefore, poten-
tially holds promise in providing an experimental model to
dissect selective forces that drive the switch from CCR5 to
CXCR4 use. To establish the utility of the model, however, it
will be necessary to demonstrate that phenotypic switching
occurs in other SHIVSF162P3N-infected macaques. Further-
more, genotypic and functional studies of additional switch
variants should reveal whether there is only one mutational
pathway for evolution toward CXCR4 usage of SHIVSF162P3,
or, alternatively, if there are multiple pathways, to identify
common envelope properties associated with the switch. To
address these issues, six more macaques were inoculated in-
travenously with the virus. Three of the six macaques were
administered the anti-CD8 antibody cM-T807 at the time of
virus inoculation to dampen the host immune response and
promote virus replication.
All six inoculated animals were infected, with peak viremia
of 106to 108RNA copies/ml plasma at 2 weeks postinfection
(wpi). Virus replication remained high during the course of
infection, with levels that were 1 log higher in the CD8-de-
pleted macaques than in those not receiving antibody treat-
ment (Fig. 1). The exception was AL63 in the no-treatment
group. This macaque established a set point that was 1 to 2 logs
lower than the set points seen with the others and was the only
animal that seroconverted (data not shown). For the three
macaques in the no-treatment group (Fig. 1A), peripheral
CD4-positive (CD4?) T-cell counts in DE86 recovered to
within the normal range after a period of transient acute loss
and fluctuated thereafter. The loss was more progressive for
macaques AL63 and BT78 but with levels stabilizing at 8 to 10
wpi. BT78 and DE86 developed clinical signs consistent with
simian AIDS (SAIDS) and were euthanized at 13 and 12 wpi,
respectively, with circulating CD4?T-cell counts greater than
200 cells/?l of blood. For the three macaques in the CD8-
depleted group, partial restoration of peripheral CD8?T cells
was seen only in macaque CC39 (Fig. 1B). All three animals,
however, showed substantial CD4?T-cell rebound after a pe-
riod of transient loss. But while the CD4?T-cell count fluctu-
ated for macaque CC39, precipitous decline followed in CA28
and CB32 and was accompanied by a spike in viremia in ma-
caque CA28 but not in CB32 at 11 wpi. The three macaques in
the CD8-depleted group all developed clinical signs consistent
with SAIDS. Notably, at time of death (15 wpi), no circulating
CD4?T cells could be detected in macaque CA28. Thus, two
of the three macaques (BT78 and DE86) infected in the ab-
sence and all three macaques (CA28, CB32, and CC39) in-
fected in the presence of the anti-CD8 monoclonal antibody
showed a rapid-progressor phenotype that was characterized
by sustained high levels of virus replication (106to 107RNA
copies/ml plasma), weak antiviral antibody response, and early
onset of SAIDS.
Analysis of the percentage of CD4?T cells in tissue com-
partments of the two macaques (BT78 and DE86) in the no-
treatment group at necropsy showed substantial preservation
of this target cell population in lymph nodes (?40%) but not
in the lamina propria of the gut (?5%) (Fig. 1A). For the three
CD8-depleted rapid progressors, lymphoid CD4?T cells could
* Corresponding author. Mailing address: Aaron Diamond AIDS Re-
search Center, The Rockefeller University, 455 First Ave., 7th Floor, New
York, NY 10016. Phone: (212) 448-5080. Fax: (212) 448-5158. E-mail:
?Published ahead of print on 2 April 2008.
still be detected in CB32 and CC39 but not in CA28 at the time
of death (Fig. 1B). This pattern of massive CD4?T-cell loss in
lymph nodes of CA28 where CXCR4?CD4?naı ¨ve and mem-
ory T cells were enriched was reminiscent of that seen in
macaque BR24 (5) and X4 SHIV-infected macaques (4),
prompting us to examine the coreceptor usage of viruses re-
covered from this animal.
Results showed that viruses recovered from the plasma of
FIG. 1. Virologic and immunologic measurements in R5 SHIVSF162P3N-infected RMs. Results for viral load, absolute peripheral CD4?T counts, and
counts are also shown in Fig. 1B to illustrate the extent and duration of CD8?T-cell depletion. Tissues analyzed at the time of necropsy included inguinal
(Ing), iliac (Ili), mesenteric (Mes), and colonic (Col) lymph nodes (LNs), as well as lamina propria lymphocytes (LPL) from the jejunum. For tissue
measurements, baseline values generated from three uninfected macaques are shown for reference, with error bars indicating standard deviations of the
means. The greater percentage of CD4 in lymph nodes of CB32 than in those of the controls was due to sustained CD8 depletion in this macaque. ?,
time of euthanasia.
5654 NOTESJ. VIROL.
CA28 (designated SHIVCA28NP) infected both CCR5- and
CXCR4-bearing U87.CD4 target cells but with better growth
in the latter, as indicated by an inverse ratio of R5/X4 repli-
cation (Fig. 2A). In contrast, viruses recovered from lymphoid
tissues (designated SHIVCA28NL), in similarity to the results
seen with the X4 variant SHIVBR24Nin macaque BR24,
showed a clear preference for CXCR4. Entry-blocking exper-
iments with coreceptor inhibitors in TZM-bl cells confirmed
the X4 coreceptor preference of the two viruses. Replication of
SHIVCA28NPwas efficiently blocked by the CCR5 antagonist
TAK-779 (?70% inhibition at 1 ?M) but could also be inhib-
ited by the CXCR4 inhibitor AMD3100 (?20% inhibition)
(Fig. 2B). In contrast, AMD3100 blocked ?90% of replication
of the lymph node-derived virus SHIVCA28NL, with little or no
inhibition seen with TAK-779. Thus, while the plasma virus
from macaque CA28 displayed dual/mixed tropism, the lymph
node virus appeared to be specific for CXCR4.
We sequenced the envelope V3 domain of SHIVCA28NPand
SHIVCA28NLand compared it to that of the inoculating virus
SHIVSF162P3Nto identify genetic changes that confer CXCR4
usage. All SHIVCA28NLEnv clones sequenced (16 in total)
harbored the same five amino acid changes in the V3 loop
compared to the sequence of the inoculating strain, which is
comprised of two major variants (162P3N-1 and 162P3N-2)
(Fig. 3A) and several minor variants (5). These amino acid
changes included a threonine-to-histidine change at position
13 and mutations of the GPG sequence at the crown of the
loop to RRW, which increased the net positive charge of this
domain. Furthermore, the alanine residue immediately down-
stream of the crown loop sequence was deleted in SHIVCA28NL.
In contrast, clonal analysis indicated that virus SHIVCA28NPre-
covered from plasma comprised a mixture of variants. A total of
15 of 19 clones sequenced showed the RRW substitutions and
alanine deletion in the V3 loop, with 4 having sequences similar
to that of the inoculating R5 strain.
To investigate whether the changes in the V3 loop of
SHIVCA28NLdictate CXCR4 usage, recombinant EnvP3N
(CA28NLV3) in which the V3 loop of the parental R5 EnvP3N
gp160 was replaced with that of SHIVCA28NLwas constructed.
In single-round infectivity assays, EnvCA28NL gp160 medi-
ated efficient entry into CXCR4-expressing U87.CD4 cells but
with some residual infectivity of CCR5-expressing cells as well
(Fig. 3B). In contrast, EnvP3N(CA28NLV3) mediated entry
only into CXCR4-expressing indicator cells, albeit with lower
efficiency compared to that of EnvCA28NL. These results il-
lustrate that V3 mutations are necessary for coreceptor switch-
ing in macaque CA28 but that changes in other regions, such as
those in the gp120 V1/V2 and C4 and gp41 domains known to
FIG. 2. Coreceptor
(A) CXCR4 and CCR5 usage of the inoculating virus SHIVSF162P3N
(P3N), the X4 variant SHIVBR24Nfrom macaque BR24 (BR24N), and
viruses recovered from plasma (CA28NP) and lymph node (CA28NL) of
macaque CA28 at end stage disease was determined by relative replica-
tion of the SHIV viruses on U87.CD4.CCR5 and U87.CD4.CXCR4 cells.
Data are expressed as the ratio of R5:X4 replication based on p27 antigen
production in the two target cell lines after 6 to 7 days of culture.
(B) Blocking of SHIV virus entry into TZM-bl cells with 1 ?M CXCR4-
and CCR5-specific inhibitors AMD3100 and TAK-779. Error bars indi-
cate standard errors of results obtained in experiments using triplicate
wells. Results shown are representative of at least two independent ex-
usage of SHIVCA28NP
FIG. 3. (A) Comparison of V3 loop sequence of SHIVSF162P3N, SHIVCA28NP, and SHIVCA28NL. Dashes denote similarities in sequence, gaps are
indicated as dots, and the net positive charges of this region are shown on the right. The numbers in parentheses indicate the number of clones with the
indicated sequence referenced to the total number of clones sequenced. (B) Entry into U87.CD4.CCR5 (black bars) and U87.CD4.CXCR4 (white bars)
of luciferase reporter viruses expressing the envelope of SHIVSF162P3N(EnvP3N), SHIVBR24N(EnvBR24N), SHIVCA28NL(EnvCA28NL), and V3 loop
recombinant EnvP3N(CA28NLV3). All data represent means ? standard errors of the means of the results of triplicate experiments.
VOL. 82, 2008NOTES5655
modulate receptor binding and fusion (2, 3, 6, 9–11), likely
contribute to optimize SHIVCA28NLentry efficiency. Signifi-
cantly, the V3 mutations that conferred CXCR4 usage to
SHIVCA28NL differed from those previously reported for
SHIVBR24N, which included insertions of two basic amino ac-
ids (histidine and arginine) immediately upstream of the crown
of the V3 loop (5).
We demonstrated X4 virus evolution and emergence in an-
other R5 SHIVSF162P3N-infected RM in this study. Functional
data showed that while the SHIVCA28NPvirus circulating in
macaque CA28 at the time of death was composed of a mixture
of CCR5- and CXCR4-using viruses, the SHIVCA28NLvirus
recovered from lymph nodes used CXCR4 for entry almost
exclusively. Mutations in the V3 loop of SHIVCA28NLwere
largely responsible for the change in coreceptor preference but
differed from those previously reported for coreceptor switching
in macaque BR24 (5), indicating that there is more than one
pathway for CCR5-to-CXCR4 evolution of the R5 SHIVSF162P3N
isolate. Both BR24 and CA28 rapidly progressed to disease with
high viral load and in the absence of seroconversion. Further-
more, in similarity to the results seen with macaque BR24, track-
following rather than preceding the onset of precipitous CD4?
T-cell decline, and the emerging variant was highly sensitive to
tempos and conditions of X4 evolution and emergence in the two
macaques points to a common mechanistic cause. Our findings
obtained with macaque CA28 confirm that the SHIVSF162P3N
isolate has an intrinsic tendency to mutate toward CXCR4 usage,
providing the tool for an experimental animal model for study of
the process and requisites for coreceptor switching.
Nucleotide sequence accession numbers. The predicted
gp160 amino acid sequences of EnvBR24N and EnvCA28NL
have been deposited in GenBank (accession numbers EU564103
and EU564104, respectively).
We thank Lisa Chakrabarti and Allen Mayer for critical reading of
the manuscript and Keith Reimann and Centocor for the anti-human
CD8 monoclonal antibody (CM-T807). AMD3100, TAK-779, and
TZM-bl cells and the U87.CD4 indicator cell line were obtained
through the NIH AIDS Research and Reference Reagent Program.
This work was supported by National Institutes of Health (NIH)
grants R01 AI46980 and R37AI41945.
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