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Journal of Virology, September 1998, p. 7603-7608, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Continued Utilization of CCR5 Coreceptor by a Newly Derived
T-Cell Line-Adapted Isolate of Human Immunodeficiency Virus Type
1
Kathryn E.
Follis,
Meg
Trahey,
Rachel A.
LaCasse, and
Jack H.
Nunberg*
Montana Biotechnology Center, The University
of Montana, Missoula, Montana 59812
Received 3 April 1998/Accepted 8 June 1998
 |
ABSTRACT |
The differential use of CC chemokine receptor 5 (CCR5) and CXC
chemokine receptor 4 (CXCR4) may be intimately involved in the
transmission and progression of human immunodeficiency virus infection.
Changes in coreceptor utilization have also been noted upon adaptation
of primary isolates (PI) to growth in established T-cell lines. All of
the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4
but not CCR5. This observation had been suggested as an explanation for
the sensitivity of TCLA, but not PI, viruses to neutralization by
recombinant gp120 antisera and V3-directed monoclonal antibodies, but
recent studies have shown coreceptor utilization to be independent of
neutralization sensitivity. Here we describe a newly isolated TCLA
virus that is sensitive to neutralization but continues to utilize both
CXCR4 and CCR5 for infection. This finding further divorces coreceptor
specificity from neutralization sensitivity and from certain changes in
cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of
CCR5 expression in the FDA/H9 T-cell line suggests that the interaction
between envelope protein and coreceptor may be mediated by multiple
weak interactions along a diffuse surface.
| |
TEXT |
The discovery of cellular molecules
that act as coreceptors in conjunction with CD4 to mediate the binding
and entry of human immunodeficiency virus type 1 (HIV-1) has provided a
new perspective from which to approach questions of HIV-1 biology and
pathogenesis. The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) by primary isolates
of HIV-1 throughout infection may have important implications for virus
transmission and disease progression. HIV-1 infection first manifests
as an acute viremic episode, typically involving a homogeneous
outgrowth of monocytotropic, non-syncytium-inducing (NSI) viruses
(53, 62) that utilize CCR5 as a coreceptor. Although the
initial events in virus transmission are largely inaccessible to
analysis, cells of the monocyte-macrophage lineage are believed to
provide a portal for primary infection and a specific filter for
monocytotropic NSI viruses (20). Persons lacking a
functional CCR5 coreceptor are resistant to the establishment of HIV-1
infection (8, 28, 43).
Viruses that utilize the CXCR4 coreceptor evolve over the course of
infection (61). These T-lymphocytotropic viruses no longer
infect monocyte-derived macrophages (45, 46) but generally continue to utilize CCR5 in addition to CXCR4 (7).
Endogenous production of CCR5-specific chemokines may provide the
selective pressure for this broadening in coreceptor use
(44). Importantly, the emergence of
dual-coreceptor-utilizing syncytium-inducing (SI) viruses in a
proportion of infected persons is prognostic for the development of
clinical AIDS (50).
In contrast to primary isolate (PI) viruses, the commonly used
laboratory isolates of HIV-1 utilize only CXCR4 as a coreceptor (1, 2, 6, 10, 12, 13, 27, 47). These isolates have been
adapted to persistent growth in T-cell lines, and the loss of their
ability to utilize CCR5 is perhaps understandable in that most T-cell
lines express CXCR4 but not CCR5 (1, 15).
Coincident with changes in coreceptor utilization and cell tropism upon
adaptation are changes in neutralization sensitivity. In contrast to PI
viruses, T-cell line-adapted (TCLA) viruses are generally sensitive to
neutralization by appropriate antibodies directed to the third variable
loop (V3) of envelope surface protein gp120 (42, 55). In
addition, PI viruses are entirely refractory to neutralization by
recombinant HIV envelope protein gp120 (rgp120) antisera that potently
neutralize related TCLA viruses (31, 55, 56). The unique
ability of PI viruses to utilize CCR5 had been suggested as a basis for
the ability of these viruses to escape neutralization, but recent
reports have shown that PI viruses remain refractory to neutralization,
regardless of the specific coreceptor utilized (27, 32, 52).
As part of our studies to define the relationship between changes in
coreceptor utilization and virus phenotype, we isolated a TCLA
derivative of molecularly cloned SI primary virus ACH320.2A.1.2 (21, 22). Although the TCLA virus was now able to infect
T-cell lines and was sensitive to antibody-mediated virus
neutralization, this virus continued to utilize both CCR5 and CXCR4
coreceptors. The intact capacity of this TCLA virus to utilize CCR5
suggests that changes in coreceptor utilization are neither associated with changes in neutralization sensitivity nor required for changes in
cell tropism.
Adaptation of a molecularly cloned SI primary virus.
The
infectious molecularly cloned provirus ACH320.2A.1.2 was isolated from
a biologically cloned SI PI obtained from a member of the
Amsterdam Cohort 9 weeks after seroconversion (21, 22). The
ACH320.2A.1.2 plasmid was obtained from Hanneke Schuitemaker (Central
Laboratory of the Netherlands Red Cross) through the NIBSC AIDS
Reagent Project (United Kingdom). Virus (320SI) was regenerated by
electroporation and expansion in phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). Supernatants containing high levels of 320SI were used in efforts to adapt this PI virus to growth
in an established T-cell line. As in previous studies (55), we utilized the Food and Drug Administration (FDA) isolate of the H9
cell line (37) because of its increased ability, relative to
other H9 cell lines, to manifest a cytopathic effect upon infection. By
other measures, FDA/H9 cells behave similarly to other H9 cell lines
(unpublished data).
In contrast to our previous experience obtained by using a biological
isolate of another primary SI virus, ACH168.10 (50, 55),
several long-term attempts to obtain infection of FDA/H9 cells by 320SI
were without success. In an effort to increase the genetic complexity
of the virus population, 320SI virus infection was first established in
the permissive human T-cell leukemia virus type 1 (HTLV-1)-transformed MT4 T-cell line (25). Continued cocultivation of these 320SI-infected MT4 cell cultures with FDA/H9 cells yielded immunofluorescence evidence of infection of the FDA/H9
cells after 4 weeks, and a low level of FDA/H9 cell infection was
subsequently obtained on passage of the culture supernatant onto naive
FDA/H9 cells. Complete and persistent infection of this culture was
obtained in an additional 3 weeks, and the TCLA virus produced
was designated 320SI-C3. Subsequent low-multiplicity passages onto fresh FDA/H9 cells yielded virus populations
320SI-C3.1 through -C3.3 over the course of 5 months. These
TCLA viruses readily infect FDA/H9 and H9 cells and yield virus titers
comparable to those of prototypic TCLA viruses.
Coreceptor utilization by the pedigreed PI and TCLA viruses.
Based on the SI phenotype of the parental 320SI virus, we anticipated
that this virus would utilize both CCR5 and CXCR4 as coreceptors for
infection. Similarly, we anticipated that the TCLA 320SI-C3 virus would
have lost the ability to utilize CCR5, as all of the TCLA isolates
analyzed to date utilize CXCR4 but not CCR5 (1, 2, 6, 10, 12,
13, 27, 47). To assess coreceptor utilization, cell culture
supernatants of infected PBLs (320SI) or FDA/H9 cells (320SI-C3) were
titrated for infectivity in U87 human glioma cells expressing CD4 and
either CCR5 or CXCR4 (23, 27). After 2 days of incubation,
cell monolayers were fixed with methanol-acetone and
immunochemically stained by using HIV/IG (38), an
anti-human ABC kit (Biomeda Corp.), and a diaminobenzamidine substrate.
Contrary to expectations based on other TCLA viruses, relative
utilization of CCR5 and CXCR4 remained unchanged upon adaptation
(Fig.
1). The TCLA 320SI-C3 virus continued to
utilize CCR5, despite
adaptation and growth in a T-cell line that
nominally does not
express CCR5. Flow cytometric analysis using
CCR5-directed monoclonal
antibody (MAb) 2D7 (
58) was able to
detect CCR5 expression on
PBLs and on the PM-1 T-cell line
(
29) but was unable to detect
its expression on FDA/H9 cells
(data not shown).
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FIG. 1.
Coreceptor utilization by pedigreed 320SI viruses.
U87-CD4 cells expressing CCR5 ( ) or CXCR4 ( ) were infected with
serial dilutions of the following viruses: 320SI from PBL culture
supernatant and 320SI-C3 and 320SI-C3.3 from FDA/H9 cell culture
supernatant. Foci were quantitated 2 days later as described in the
text. Relative utilization of CCR5 and CXCR4 was judged by the relative
titer on the respective coreceptor-expressing cells.
Coreceptor-specific titer was determined on the linear portion of the
virus titration curve.
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|
We wanted to confirm that this unique pattern of dual coreceptor use by
a TCLA virus was stable through additional passage
in FDA/H9 cells. We
examined coreceptor utilization by the repeatedly
passaged 320SI-C3.3
virus. This virus was also indistinguishable
from the original PI virus
320SI in coreceptor specificity (Fig.
1). Thus, dual use of CCR5 and
CXCR4 appears to be a stable property
of the TCLA 320SI-C3 virus.
The adaptation procedure used here to obtain 320SI-C3 differs from
that used previously to isolate a TCLA derivative of another
SI primary
virus, ACH168.10 (
50,
55). This biological isolate
was able
to directly establish infection in the FDA/H9 cell line,
albeit
over the course of 4 to 12 weeks, without the need for
expansion in MT4
cells. Although expansion in permissive MT4 cells
may have been
important in generating sequence diversity in the
molecularly cloned
320SI virus population, it was also conceivable
that this passage
history affected the adaptation process and
coreceptor preference of
the ultimate TCLA virus.
To address this possibility, we repeated the adaptation of ACH168.10
(168P) by using an MT4 cell line intermediate. The 168M
virus derived
from the MT4 cell culture retained PI virus phenotypes,
including
dual-coreceptor utilization and the lack of measurable
FDA/H9 cell
tropism, as did the MT4 cell line-expanded 320SI virus
(data not
shown). This observation supports the contention that
MT4 cells are
permissive and nonselective towards SI viruses,
serving here simply to
broaden the quasispecies distribution of
the molecularly cloned 320SI
virus. Following cocultivation and
adaptation of 168M to persistent
growth in FDA/H9 cells, the newly
derived TCLA virus 168MC2 lost the
ability to utilize CCR5 (Fig.
2), as had
the original TCLA 168C virus that was directly adapted
to growth in
FDA/H9 cells (
27,
55). Thus, the retention of
dual-coreceptor use by the TCLA 320SI-C3 virus is not a reflection
of
its passage through MT4 cells prior to adaptation to growth
in the
FDA/H9 cell line. Rather, different viruses appear to respond
differently to selection for growth in T-cell lines.
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FIG. 2.
Coreceptor utilization by TCLA virus 168MC2 adapted
through an MT4 cell line intermediate. U87-CD4 cells expressing CCR5
() or CXCR4 () were infected with serial dilutions of the
following viruses: 168P from PBL culture supernatant (27,
55), 168MC2 from FDA/H9 cell culture supernatant following
expansion in an MT4 cell culture, and 168C from FDA/H9 cell culture
supernatant following direct adaptation to FDA/H9 cells (27,
55).
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Neutralization sensitivity of the TCLA 320SI-C3 virus.
Previous studies had demonstrated that adaptation to persistent growth
in established T-cell lines renders the TCLA virus sensitive to
neutralization by a broad range of reagents (33, 55).
Similarly, TCLA virus 320SI-C3 demonstrated de novo sensitivity to
V3-directed MAbs 50.1, 58.2, and 59.1 (Fig.
3a to c, respectively) (54).
The core epitopes of the latter two MAbs have been mapped and are
present in the known amino acid sequence of 320SI (IGPGRAF and GPGRAF,
respectively [19, 54]); the core epitope of MAb 50.1 has also been determined crystallographically (RIHIG
[39]) and differs from that present in 320SI (GIHIG).
The 320SI-C3 virus was also newly sensitive to CD4 binding
domain-directed MAb IgG1 b12 (5), to CD4 immunoadhesin
(CD4-Ig) (48), and to HIVIG (Fig. 3d to f, respectively).
Thus, as in previous studies, increased sensitivity to virus
neutralization appeared coordinately with adaptation to growth in
established T-cell lines.
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FIG. 3.
Neutralization sensitivity of 320SI (PI) and 320SI-C3
(TCLA) in U87-CD4 cell lines expressing the CCR5 or CXCR4 coreceptor.
Virus stocks of 320SI (, ) and 320SI-C3 ( ,  ) comprising
cell culture supernatants (from PBLs and FDA/H9 cells, respectively)
were standardized to yield a submaximal number of foci of infection on
U87-CD4-CCR5 (, ) or U87-CD4-CXCR4 (, ) cells. The virus
was incubated with the indicated reagents for 1 h prior to
infection (a to f).
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|
Because of the dual-coreceptor use by these PI and TCLA viruses, we
were able to investigate neutralization sensitivity as
a function of
coreceptor use (Fig.
3). In all cases, neutralization
sensitivity was
independent of the coreceptor utilized for infection
of U87-CD4 cells.
We had previously demonstrated this for PI virus
ACH168.10
(
27); we can now extend this conclusion to a TCLA
virus. Furthermore, not only is neutralization sensitivity independent
of the process of utilizing a specific coreceptor, it is also
independent of the changes in coreceptor specificity that often
accompany adaptation to growth in T-cell lines.
DNA sequence analysis of the 320SI-C3 envelope gene.
In our
previous analysis of the adaptation of ACH168.10 (168P), we defined
three adaptation-associated amino acid changes within the envelope
protein of TCLA virus 168C: I166R in V2, I282N in C2, and G318R in V3
(55). For the present study, we wished to determine whether
similar amino acid changes had arisen during adaptation of the 320SI
virus. Proviral DNA from FDA/H9 cells infected with 320SI-C3 was
amplified by using envelope-specific primers (envA and envN
[18]) and high-fidelity XL PCR (PE Applied Biosystems)
(27), and the DNA sequence of the envelope gene was compared
with those of ACH320.2A.1.2 (22) and 320SI. Two amino acid
changes in gp120 were noted (I166K and H317R (GenBank accession
no. AF069524). The I166K change in the V2 region is similar to the
I166R change in the unrelated 168C envelope. We do not know
whether the change is fortuitous or whether this common amino acid
change points to a common function. The H317R change is
located within the crown of the V3 loop
(KGIHIGPGRAF to KGIRIGPGRAF) but
distant from the G318R change in the 168C envelope
(GRAFYTTRQII). The increased positive charge
in the V3 loop is consistent with similar trends in other PI and TCLA
viruses (9, 17, 55). In the case of the 320SI-C3 virus,
however, the increased positive charge may be associated with
adaptation and neutralization sensitivity but is not associated with
altered coreceptor use.
In summary, our finding of this unique TCLA virus that is now sensitive
to neutralization but continues to utilize both CCR5
and CXCR4 extends
our earlier observations divorcing coreceptor
utilization and
sensitivity to neutralization. Not only are PI
viruses resistant to
neutralization regardless of whether CCR5
or CXCR4 is used in
infection, but changes in the ability to utilize
these coreceptors are
not required for the acquisition of neutralization
sensitivity in the
TCLA virus. Furthermore, the TCLA virus remains
sensitive to
neutralization regardless of the coreceptor utilized
for infection.
Our findings also divorce changes in coreceptor utilization and
adaptation to growth in established T-cell lines. Clearly,
the
often-observed loss of the ability of TCLA viruses to utilize
CCR5 is
not necessary for adaptation. It is possible, however,
that adaptation
might derive from more subtle changes in the virus's
interaction with
CXCR4. Initial studies to examine the sensitivity
to inhibition by the
CXCR4 ligand SDF-1 (
4,
34), however,
did not yield a
consistent pattern: whereas 320SI-C3 showed increased
sensitivity
to inhibition by SDF-1, 168C showed decreased sensitivity
(data not
shown). Thus, the relationship between coreceptor utilization
and
adaptation to growth in T-cell lines remains elusive. Appropriate
coreceptor expression may be necessary for infection, but it is
by no
means sufficient, as restrictions to productive infection
can exist at
multiple levels in the viral life cycle (
16,
59,
60).
Our observations that these phenotypically distinct viruses can
continue to utilize both coreceptors and that the capacity
to utilize
CCR5 is retained despite the apparent lack of the CCR5
coreceptor on
FDA/H9 cells suggest that the structural requirements
for specific
coreceptor binding are relatively minimal. In fact,
multiple studies to
define critical sites for envelope-coreceptor
interaction have not
yielded entirely consistent generalizations
(
3,
11,
26,
35,
36,
40,
41,
49). The variable
and diffuse nature of functional
envelope-coreceptor pairings
is consistent with an interaction surface
comprising multiple
weak contacts. We suggest that the free energy of
coreceptor binding
may derive in part from the entropic contribution of
the initial
binding to CD4 in localizing the envelope-coreceptor
interaction
to two dimensions. Subsequent interactions are driven both
by
conformational changes induced within the envelope protein complex
upon CD4 binding (
51,
57) and by proximity on the membrane
but ultimately involve multiple weak contacts over a variable
surface
of envelope-coreceptor interaction. The observed relationship
between
infectivity by PI viruses and cell surface CD4 density
is consistent
with this model (
24).
In certain instances, the entropic contributions of CD4 binding may be
less significant to the overall free energy of envelope
protein
binding, as in certain human and simian immunodeficiency
virus isolates
that are able to interact directly with a coreceptor
to allow
CD4-independent infection (
14,
15,
30). This capability
may
derive from an envelope protein conformation that more closely
approximates that induced by CD4 binding, or perhaps from other
enhancements in the envelope-coreceptor interaction.
To the extent that the envelope-coreceptor interaction is diffuse and
that specificity is determined by the sum of many weak
contacts, the
effects of specific changes in either protein on
overall binding may
not be readily predictable. The changes in
the 320SI-C3 envelope
protein that determine cell tropism appear
not to perturb significantly
the envelope-CCR5 binding surface,
whereas those in the 168C envelope
protein preclude effective
interaction. Perhaps subtle changes in
envelope-coreceptor interaction
underlie the observed changes in cell
tropism and neutralization
sensitivity that define adaptation. At the
current level of analysis,
however, these fundamental viral phenotypes
appear to be independent
of specific coreceptor utilization.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH AREA grant AI41165 to J.H.N.
We thank Jim Rusche and Carolyn Muermann (Repligen Corp.) for gifts of
V3-directed MAbs 50.1, 58.2, and 59.1. Additional reagents were kindly
provided by Steve Chamow (Genentech, Inc.), Dan Littman (HHMI, NYU
Medical Center), Ian Clark-Lewis (University of British Columbia),
Susan Zolla-Pazner (NYU Medical Center), and Fred Prince (NY Blood
Center). The following MAbs were obtained through the AIDS Research and
Reference Reagent Program, NIH, NIAID: 2D7 from LeukoSite, Inc.,
and IgG1 b12 from Dennis Burton and Carlos Barbas. The
infectious molecularly cloned provirus ACH320.2A.1.2 was obtained through the NIBSC AIDS Reagent Project (United Kingdom) from
Hanneke Schuitemaker. We thank Dave Holley for technical assistance,
Joan Strange for DNA sequencing services (The University of Montana M. J. Murdock Molecular Biology Facility), Richard Field
(Department of Chemistry) for valuable discussions, and Ed Walker and
Linda Griggs (Ribi ImmunoChem Research, Inc.) for flow
cytometry.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Montana
Biotechnology Center, The University of Montana, Missoula, MT 59812. Phone: (406) 243-6421. Fax: (406) 243-6425. E-mail:
nunberg{at}selway.umt.edu.
| |
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Journal of Virology, September 1998, p. 7603-7608, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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