Anisha Misra,
aEmile Gleeson,
aWeiming Wang,
bChaobaihui Ye,
bPaul Zhou,
bJason T. Kimata
aaDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
bUnit of Antiviral Immunity and Genetic Therapy, Institute Pasteur of Shanghai-Chinese Academy of Sciences,
Shanghai, China
ABSTRACT
In previous studies, we demonstrated that single-chain variable
frag-ments (scFvs) from human immunodeficiency virus (HIV) Env monoclonal
anti-bodies act as entry inhibitors when tethered to the surface of target cells by a
glycosyl-phosphatidylinositol (GPI) anchor. Interestingly, even if a virus escapes
inhi-bition at entry, its replication is ultimately controlled. We hypothesized that in
addi-tion to funcaddi-tioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env
in an infected cell, thereby interfering with the infectivity of newly produced virions.
Here, we show that expression of the anti-HIV Env GPI-scFvs in virus-producing cells
reduced the release of HIV from cells 5- to 22-fold, and infectivity of the virions that
were released was inhibited by 74% to 99%. Additionally, anti-HIV Env GPI-scFv X5
inhibited virion production and infectivity after latency reactivation and blocked
transmitter/founder virus production and infectivity in primary CD4
⫹T cells. In
con-trast, simian immunodeficiency virus (SIV) production and infectivity were not
af-fected by the anti-HIV Env GPI-scFvs. Loss of infectivity of HIV was associated with a
reduction in the amount of virion-associated Env gp120. Interestingly, an analysis of
Env expression in cell lysates demonstrated that the anti-Env GPI-scFvs interfered
with processing of Env gp160 precursors in cells. These data indicate that GPI-scFvs
can inhibit Env processing and function, thereby restricting production and
infectiv-ity of newly synthesized HIV. Anti-Env GPI-scFvs therefore appear to be unique
anti-HIV molecules as they derive their potent inhibitory activity by interfering with both
early (receptor binding/entry) and late (Env processing and incorporation into
viri-ons) stages of the HIV life cycle.
IMPORTANCE
The restoration of immune function and persistence of CD4
⫹T cells
in HIV-1-infected individuals without antiretroviral therapy requires a way to increase
resistance of CD4
⫹T cells to infection by both R5- and X4-tropic HIV-1. Previously,
we reported that anchoring anti-HIV-1 single-chain variable fragments (scFvs) via
glycosyl-phosphatidylinositol (GPI) to the surface of permissive cells conferred a high
level of resistance to HIV-1 variants at the level of entry. Here, we report that
anti-HIV GPI-scFvs also derive their potent antiviral activity in part by blocking anti-HIV
pro-duction and Env processing, which consequently inhibits viral infectivity even in
pri-mary infection models. Thus, we conclude that GPI-anchored anti-HIV scFvs derive
their potent blocking activity of HIV replication by interfering with successive stages
of the viral life cycle. They may be effectively used in genetic intervention of HIV-1
infection.
KEYWORDS
envelope protein, antiviral agents, entry inhibitor, human
immunodeficiency virus, infectivity, neutralizing antibodies, scFv
Received29 November 2017Accepted2
January 2018
Accepted manuscript posted online10
January 2018
CitationMisra A, Gleeson E, Wang W, Ye C,
Zhou P, Kimata JT. 2018. Glycosyl-phosphatidylinositol-anchored anti-HIV Env single-chain variable fragments interfere with HIV-1 Env processing and viral infectivity. J Virol 92:e02080-17.https://doi.org/10.1128/JVI .02080-17.
EditorFrank Kirchhoff, Ulm University Medical
Center
Copyright© 2018 American Society for
Microbiology.All Rights Reserved. Address correspondence to Jason T. Kimata, [email protected].
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T
he human immunodeficiency virus (HIV) Env protein mediates infection of CD4
⫹T
cells through both cell-free and cell-to-cell mechanisms. Both mechanisms require
the assembly of infectious particles (1). However, cell-to-cell transmission has been
found to be 100 to 1,000 times more efficient than cell-free infection (2–5). Cell-to-cell
transmission requires the formation of virological synapses between the infected and
target cells (6–8), thus allowing the virus to bypass various kinetic and immunological
barriers (9, 10). Although it is difficult to distinguish the individual contributions of each
mode of infection to the spread of HIV, a recent study suggests that cell-to-cell
transmission may account for 60% of infection, reduce the rate of virus generation, and
increase viral fitness by 3.9-fold (11). These data support previous findings in the BLT
humanized mouse model in which preventing the formation of virological synapses by
blocking the release of HIV-1-infected T cells from lymph nodes into naive lymph nodes
greatly limited HIV-1 systemic infection (12). Importantly, these studies show that
inhibitors of HIV must neutralize both modes of infection in order to effectively block
viral replication and protect CD4
⫹T cells from infection.
The activities of most antiretroviral drugs and neutralizing antibodies are most
effective at blocking cell-free HIV, whereas cell-to-cell transmission of HIV may be less
susceptible to inhibition (3, 10, 13). In T cell cocultures, neutralization is observed only
when infected T cells are pretreated with antibodies before coculturing with uninfected
T cells (3, 9, 10, 12). Due to different experimental approaches as well as differential
activity of neutralizing antibodies on the viral life cycle, there are conflicting data on
whether neutralizing antibodies inhibit cell-to-cell transmission of HIV. One study
observed that both anti-gp41 and anti-gp120 antibodies can block synaptic spread of
HIV (14), but another showed that only anti-gp41 antibodies are capable of blocking
synaptic infection (15). Moreover, other studies have shown that since anti-gp41
antibodies such as 2F5 and 4E10 are unable to block gp120-CD4 engagement, they do
not inhibit synapse formation or virion transfer (3, 16), but they are capable of blocking
any resulting infection (16, 17). However, HIV-1 bound by antibodies such as 2F5, 4E10,
and 10E8 can be captured by dendritic cells (DCs) and infect CD4 T cells, but infection
is inhibited when the virion is bound to b12, NIH45-46, and VRC01 antibodies (18, 19).
In-depth studies where a panel of 16 broadly neutralizing antibodies were tested
against 11 different HIV-1 strains during both cell-free and cell-to-cell transmission
attempt to explain the conflicting data previously published by concluding that the
efficacy of inhibition by these neutralizing antibodies is not only strain and epitope
dependent but also depends on the specific inhibitory steps during the entry process
(20).
HIV utilizes lipid rafts of the plasma membrane as gateways for entry into T cells and
macrophages (21), as well as budding of newly assembled virions (21–23). Lipid rafts are
biophysically and biochemically distinct regions of the plasma membrane that maintain
an ordered structure and are rich in cholesterol and sphingomyelin (24). It is known that
CD4, the receptor for HIV-1 entry (25, 26), and the HIV Env protein colocalize with
polarized raft markers GM1 and CD59 but not with transferrin receptor, which is
excluded from lipid rafts (27). Previously, we showed that binding regions of anti-HIV
Env monoclonal antibodies, such as single-chain Fv (scFv) (28), the heavy-chain
complementarity-determining region 3 (HCDR3) of PG16, which binds a linear epitope
on the HIV-1 Env (29), llama single-chain (30) antibodies, or even the C34 peptide of the
HIV Env gp41 protein (31), can be targeted to lipid rafts by genetically linking them to
a glycosyl-phosphatidylinositol (GPI) attachment signal from decay-accelerating factor
(DAF) (32). Interestingly, when these molecules are targeted to the lipid raft using a GPI
anchor, they exhibited potent neutralizing activity, blocking entry of different HIV-1
subtypes and variants in both cell-free infection and cell-cell transmission assays
(28–31, 33). Thus, directing neutralizing molecules against HIV-1 to lipid rafts with a GPI
anchor appears to improve their capacity to protect permissive cells from infection.
Among the GPI-anchored scFvs (GPI-scFvs), X5 showed the most potent and broadly
neutralizing ability against both HIV-1 Env pseudotyped virions as well as wild-type
HIV-1 (28). The X5 antibody is thought to recognize a conserved CD4-inducible epitope
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(34). Interestingly, even during long-term
in vitro
infection experiments, GPI-X5
dem-onstrated remarkable inhibitory activity against HIV (28). These studies show the
potential of GPI-anchored scFv to neutralize virus entry and to provide long-term
protection as well as interfere with cell-to-cell transfer of HIV.
In our past studies, we attributed most of the blocking activity of anti-HIV Env
GPI-scFvs to interference with entry. However, in studies of GPI-X5, we discovered that
virus replication is ultimately controlled during long-term infections even if a small
percentage of target cells modified with GPI-X5 become infected. We hypothesized that
in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may interact with Env
in infected modified cells, thereby interfering with infectivity of newly produced virions.
In the present studies, we test this hypothesis. Our data indicate that GPI-scFvs also
have the ability to inhibit Env processing and virion incorporation in virus-producing
cells coexpressing anti-HIV-1 Env GPI-scFvs, thereby restricting production and
infec-tivity of newly synthesized HIV. We determined that these results could be
recapitu-lated with transmitter/founder (T/F) HIV-1 strains and even in infections in primary
CD4
⫹T cells and after virus is reactivated from latency. Thus, anti-HIV Env GPI-scFvs in
part also derive their potent inhibitory activity against HIV by interfering with late
stages of the virus life cycle.
RESULTS
Expression of GPI-scFvs after cotransfection into 293T cells.
Our past studies
with CD4
⫹cells stably expressing GPI-scFvs from integrated lentiviral vectors
demon-strated potent blocking of HIV-1 entry (28, 35). In order to bypass viral entry and
examine the effect of anti-HIV Env GPI-scFvs on later stages of the viral replication cycle,
we cloned the GPI-scFv fusion genes into the plasmid expression vector pcDNA3, which
was then cotransfected with HIV-1 proviruses into 293T cells. First, we assessed
expres-sion of the GPI-scFvs on the cell surface by flow cytometry. Figure 1 shows similar
high-level surface expression levels of the GPI-AB65 (anti-influenza virus hemagglutinin
[HA] control scFv vector) control and anti-HIV Env gp120 GPI-X5 and GPI-PG16
con-structs when they were cotransfected with an HIV proviral DNA clone.
Anti-Env GPI-scFvs restrict HIV-1 virion release.
To examine the effect of GPI-scFv
expression on release of HIV-1, 293T cells were cotransfected with the GPI-scFv
con-structs (control GPI-AB65 and either anti-HIV-1 Env GPI-X5 or GPI-PG16) and CXCR4- or
CCR5-tropic proviral clones of HIV-1 NL4-3 or AD8, respectively. After 48 h, p24
gagwas
measured in the supernatants by enzyme-linked immunosorbent assay (ELISA) as an
indicator of virus production. Compared to cotransfections with either the empty vector
or GPI-AB65 control, production of HIV-1 NL4-3 p24
gagwas significantly decreased 6- to
10-fold by GPI-X5 and 3.2- to 6-fold by GPI-PG16 (Fig. 2A). Similarly, anti-HIV GPI-X5 and
GPI-PG16 significantly reduced HIV-1 AD8 p24
gagproduction 4- to 5-fold and 5- to
7-fold, respectively (Fig. 2B). The data indicate that virion release is inhibited from cells
expressing anti-HIV GPI-scFvs.
FIG 1Expression levels of GPI-scFv constructs after cotransfection. 293T cells were transfected with GPI-scFv constructs and harvested, and GPI-positive cells were quantified by staining for the His-tagged hinge region by flow cytometry. SSC, side scatter.
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[image:3.585.56.356.71.198.2]Anti-Env GPI-scFvs inhibit infectivity of progeny virions.
Next, in order to assess
the infectivity of the virions released from the GPI-scFv-expressing cells, supernatants
containing equal amounts of p24
gagwere used to infect TZM-bl indicator cells, and the
relative level of infection was determined by measuring luciferase activity in lysates.
Compared to infectivity of HIV-1 NL4-3 produced from the empty vector
negative-control cells, infectivity of NL4-3 produced from cells expressing the anti-HIV Env GPI-X5
and GPI-PG16 was decreased significantly by 91-fold and 25-fold, respectively (Fig. 3A).
Relative to virus produced from the negative-control GPI-AB65 construct-expressing
cells, NL4-3 produced from GPI-X5 and GPI-PG16 was reduced by 49-fold and 14-fold,
respectively. The GPI-AB65 construct did not significantly inhibit infectivity of NL4-3.
Similar to the results obtained with HIV-1 NL4-3, GPI-AB65 had no effect on infectivity
of HIV-1 AD8. However, GPI-X5 and GPI-PG16 decreased infectivity of AD8 by 22-fold
and 4-fold, respectively (Fig. 3B). Together, these data indicate that HIV-1 produced
from cells expressing anti-HIV Env GPI-scFvs loses viral infectivity.
FIG 2Production of virions is significantly decreased by anti-HIV Env GPI-scFvs. 293T cells were cotransfected with GPI-scFvs and HIV clones NL4-3 (A) and AD8 (B). Supernatants were collected, and titers were determined by p24 ELISA. Histograms show means⫾standard deviations (n⫽3). Statistical comparisons were done by ANOVA (**,
P⬍0.01,***,P⬍0.001;****,P⬍0.0001). All results shown are representative of three independent experiments.
FIG 3Anti-HIV Env GPI-scFvs significantly reduce viral infectivity. Equal amounts of HIV p24 collected from cotransfections of NL4-3 (A) and AD8 (B) were used to infect TZM-bl cells. Histograms show means⫾standard deviations (n⫽3). Statistical comparisons were done by ANOVA (*,P⬍0.05;**,P⬍0.01,***,P⬍0.001;****,
P⬍0.0001). All results shown are representative of three independent experiments.
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[image:4.585.40.400.71.255.2] [image:4.585.42.403.521.695.2]Anti-HIV-1 Env GPI-scFvs do not inhibit SIV production or infectivity.
To assess
whether the effect of the anti-HIV-1 Env GPI-scFvs is specific for HIV-1, we cotransfected
293T cells with simian immunodeficiency virus ([SIV] SIVmne027) and either the control
GPI-AB65 or anti-HIV-1 Env GPI-X5 or GPI-PG16. SIV p27
gagproduction from cells
expressing either the control or anti-HIV-1 Env GPI-scFvs was not significantly different
from that with the vector control (Fig. 4A). Additionally, there was no difference in
infectivity levels of SIV released from the negative controls compared to the level in
cells expressing GPI-scFvs (Fig. 4B). Thus, anti-HIV-1 Env GPI-scFvs appear to specifically
block HIV-1 production and infectivity.
Anti-Env GPI-scFvs inhibit HIV envelope processing.
Since the anti-HIV-1 Env
GPI-scFvs were specific to inhibiting HIV-1, we wondered whether GPI-X5 and GPI-PG16
altered processing of Env gp160 to gp120 and gp41 when they were coexpressed with
HIV-1. Western blot analysis with anti-Env antiserum was used to determine if there
were differences in the amounts of precursor gp160 and gp120 associated with anti-HIV
Env GPI-scFv expression in cells cotransfected with GPI-scFvs and HIV-1 proviral clones.
Interestingly, when either HIV-1 NL4-3 or AD8 was cotransfected with GPI-X5, there was
less Env gp120 detected (Fig. 5A). Compared to levels in the controls, there was also a
relative decrease in Env gp160 for HIV-1 NL4-3 but not for AD8. Moreover, the ratio of
gp120/gp160 was reduced (Fig. 5B). On the other hand, GPI-PG16 had little effect on
the level of HIV-1 AD8 Env gp120 and appeared to have less of an effect on the NL4-3
Env gp120 than GPI-X5 (Fig. 5A and B). Gag p24 levels appeared to be unaffected by
expression of the GPI-scFvs. These data indicate that anti-HIV-1 Env GPI-scFvs,
partic-ularly GPI-X5, can interfere with Env processing in HIV-1-producing cells.
Anti-Env GPI-scFvs block incorporation of gp120 in HIV-1 virions.
Because Env
processing was inhibited by the anti-HIV-1 Env GPI-scFvs, we next examined whether
FIG 4Production and infectivity of SIV is unaffected by anti-HIV Env GPI-scFvs. (A) Quantification of SIV produced in the presence or absence of GPI-scFvs using an SIV p27 ELISA. (B) Relative infection levels with SIV derived in the presence or absence of the indicated GPI-scFvs. Histograms show means ⫾ standard errors of the means (n ⫽ 3). All results shown are representative of three independent experiments.on November 6, 2019 by guest
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[image:5.585.120.290.73.366.2]the block in processing of gp160 would also result in less Env gp120 associated with
virions. We therefore concentrated virus from supernatants of 293T cells cotransfected
with HIV-1 proviral clone NL4-3 or AD8 and either the control GPI-AB65 or anti-HIV-1
Env GPI-X5 or GPI-PG16. The control GPI-AB65 had some nonspecific effect on Env
incorporation into virions. However, there was a decrease in the amount of gp120
associated with virions released from cells expressing either GPI-X5 or GPI-PG16
com-pared to the level with the empty vector or GPI-AB65 negative control (Fig. 6A). This
was evident even after the amounts of gp120 were normalized to those of p24
gag(Fig. 6B).
Together, these data indicate that anti-HIV-1 Env GPI-scFvs may engage the viral Env
protein in virus-producing cells, potentially interfering with processing of Env gp160
precursor to gp120 and gp41 and reducing the amount of Env gp120 incorporated into
virions. As a consequence, there is a loss of viral infectivity.
Anti-Env GPI-scFvs inhibit T/F HIV-1 virion release and infectivity.
To examine
the effect of GPI-scFv expression on release of transmitter/founder (T/F) HIV-1, 293T
cells were cotransfected with the GPI-scFv constructs (control GPI-AB65 and either
anti-HIV-1 Env GPI-X5 or GPI-PG16) and T/F proviral clone AD17, RHPA, or THRO. After
48 h, p24
gagwas measured in the supernatants by ELISA as an indicator of virus
production. Compared to the levels for cotransfections with the empty vector control,
production of HIV-1 p24
gagwas significantly decreased in the presence of GPI-X5 and
almost completely inhibited in the presence of GPI-PG16 for all three viruses (Fig. 7A).
However, relative to the GPI-scFv control, GPI-AB65, GPI-X5 did not reduce virion
release of HIV-1 AD17 or RHPA.
To assess the infectivity of the virions released from the GPI-scFv-expressing cells,
supernatants containing equal amounts of p24
gagwere used to infect TZM-bl indicator
FIG 5Processing of Env gp160 precursors in cells is inhibited by anti-HIV Env GPI-scFvs. (A) Western blot analysis of cell lysates for gp160, gp120, p24, and actin. (B) ImageJ was used to quantify the band intensity ratio of gp120 to gp160 in cell lysates. Data shown are the averages⫾standard deviations of three experiments.***,P⬍0.001; NS, not significant.Misra et al. Journal of Virology
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[image:6.585.73.339.69.384.2]cells, and the relative level of infection was determined by measuring luciferase activity
in lysates (Fig. 7B). Insufficient amounts of virus produced in the presence of GPI-PG16
prevented testing its effect on viral infectivity. However, virions released in the
pres-ence of GPI-X5 had reduced infectivity relative to the control level. Thus, the anti-HIV
Env GPI-scFvs are also effective at reducing virion release and infectivity of T/F HIV-1
variants.
Anti-Env GPI-scFvs inhibit HIV-1 production from primary CD4
ⴙT cells.
In order
to assess the efficacy of GPI-scFvs in a more relevant cell type, CD4
⫹T cells were
isolated from peripheral blood mononuclear cells (PBMCs) and infected with T/F HIV-1
variants AD17, RHPA, and THRO and subsequently transduced with GPI-scFvs.
Trans-duction of CD4
⫹T cells was assessed by flow cytometry, which showed 68 to 75%
expression of GPI-scFvs (Fig. 8A). Since cells expressed high levels of GPI-scFvs in
infected primary CD4
⫹T cells, supernatants were collected at 14 days postinfection,
and virion production was assessed by quantifying p24
gag(Fig. 8B). The presence of
GPI-X5 completely hindered detection of T/F HIV-1 from infected CD4
⫹T cells, while
the control GPI-AB65 had no effect on virus production.
GPI-X5 inhibits HIV-1 induced from latency.
Finally, we examined whether
ex-pression of anti-HIV GPI-scFv GPI-X5 on infected cells would suppress infectious virus
after latency reactivation. For this experiment, we used ACH-2 cells which possess a
single integrated copy of the HIV-1 strain LAI that can be induced with tumor necrosis
factor alpha (TNF-
␣
) to produce infectious virus (36, 37). First, to determine if TNF-
␣
would alter GPI-scFv expression, ACH-2 cells were transduced with a GPI-AB65 or
GPI-X5 vector and exposed to increasing amounts of TNF-
␣
(1, 5, and 10 ng/ml).
Treatment with TNF-
␣
did not significantly alter the expression of GPI-scFvs on the cell
surface (Fig. 9A). Supernatants were collected after treatment with TNF-
␣
, and p24
gagwas quantified by ELISA (Fig. 9B). Even with only about 70% of the ACH-2 cells
transduced, GPI-X5 significantly inhibited the amount of virus released from ACH-2 cells
at all three concentrations of TNF-
␣
. Virions released from GPI-X5-expressing cells had
reduced infectivity compared to that of the virions released in the presence of GPI-AB65
FIG 6HIV Env gp120 associated with virions released from cells is reduced by anti-HIV Env GPI-scFvs. (A) Western blot analysis of supernatants for gp160, gp120, and p24. (B) ImageJ was used to quantify the band intensity of gp120 relative to that of p24 in virion lysates. Data shown are averages⫾standard deviations of three independent experiments.*,P⬍0.05;**,P⬍0.01,***,P⬍0.001;****,P⬍0.0001.on November 6, 2019 by guest
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[image:7.585.74.338.71.320.2]or from untransduced control cells (Fig. 9C). Thus, GPI-X5 is able to inhibit HIV-1
induced from latency.
DISCUSSION
Our past studies of anti-HIV Env GPI-anchor antibody derivatives have shown
increased potency and breadth of neutralization of HIV-1 compared to levels of their
secreted counterparts (28–31, 33). In those studies, we demonstrated that anti-HIV Env
GPI-anchored antibody derivatives severely restrict viral replication at the level of entry.
Furthermore, anti-HIV Env GPI-scFvs migrate into virological synapses and efficiently
block cell-to-cell transmission of both R5- and X4-tropic HIV (28), and, thus, they can
block both cell-free and cell-to-cell transmission of HIV. In the present study, we found
that anti-HIV Env GPI-scFvs can also inhibit viral replication at later stages of the viral
life cycle. We demonstrated that both the productivity and infectivity of the progeny
virions are lowered in the presence of anti-HIV Env GPI-scFvs and that this inhibitory
effect is specific to HIV-1. Our data also showed that anti-HIV Env GPI-scFvs inhibited
processing of the viral Env and its incorporation into newly synthesized virions. Thus,
anti-HIV GPI-scFvs derive their potent inhibitory activity against HIV-1 by interfering
with early and late stages of the viral replication cycle.
In past studies, we observed that the anti-HIV GPI-scFv GPI-X5 could inhibit viral
replication without breakthrough replication of a resistant variant virus even though
entry was not completely blocked (28). Here, using cotransfection experiments with HIV
proviruses and anti-HIV Env GPI-scFvs, we demonstrated that HIV-1 produced from cells
coexpressing the anti-HIV Env GPI-scFvs GPI-X5 and GPI-PG16 had significantly lower
levels of virus production than cells coexpressing the GPI-scFv control or
mock-FIG 7Production and infectivity of transmitter/founder virions is significantly decreased by anti-HIV Env GPI-scFvs. (A) Quantification of virus in cell supernatants. (B) Relative infectivity of T/F HIV-1 clones produced in the presence or absence of GPI-scFvs. 293T cells were cotransfected with GPI-scFvs and transmitter/founder HIV-1 clones. The amount of virus produced was determined by HIV p24 ELISA, and equal amounts of p24 from each culture were used to determine viral infectivity on TZM-bl cells. Histograms show means⫾standard deviations (n⫽3). Statistical comparisons were done by ANOVA (*,P⬍0.05; **,P⬍0.01,***,P⬍0.001;****,P⬍0.0001; NS, not significant). All results shown are representative of three independent experiments.
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[image:8.585.41.542.71.375.2]transfected control cells. Moreover, the infectivity of the released virus was decreased
relative to that of virus produced from cells expressing the GPI-scFv control, GPI-AB65.
The reduction in viral release and infectivity of progeny virions was also observed when
transmitter/founder HIV-1 strains were transfected. The effect was specific as SIV
production and infectivity were unaffected by the anti-HIV Env GPI-scFvs. These data
indicate that anti-HIV Env GPI-scFvs may engage Env during synthesis to disrupt the
production of infectious virions.
Our data support the potential for anti-HIV Env GPI-scFv to interfere with Env
synthesis. In particular, GPI-X5 reduced the appearance of gp120 but not of the
precursor protein, gp160, in cotransfected cells, suggesting that GPI-X5 engages Env
gp160 and interferes with processing. As GPI-anchored proteins are synthesized and
processed through the endoplasmic reticulum and Golgi complex (38, 39), it is
con-ceivable that GPI-X5 engages a nascent form of Env within one of the compartments
prior to cleavage by furin. While initial studies on the X5 monoclonal antibody indicated
that its binding to Env is CD4 dependent (34, 40–44), recent studies indicate that the
X5 scFv may bind Env independent of CD4 in the context of a chimeric antigen receptor
(45). Our data, including our previous studies (28), support the potential for
CD4-independent binding of membrane-anchored X5 scFv to Env. Of further interest, the
GPI-PG16 molecule has limited to no effect on Env gp160 processing although it
reduces viral infectivity, suggesting that GPI-PG16 may interact with Env later during its
synthesis. While the intracellular effect of the anti-HIV Env GPI-scFvs is reminiscent of
FIG 8GPI-X5 transduction of infected primary CD4⫹T cells inhibits production of transmitter/founder HIV-1. (A) Expression levels of GPI-scFvs on infected CD4⫹T cells. (B) HIV-1 p24 production. Primary CD4⫹T cells isolated from PBMCs were infected with three different HIV-1 transmitter/founder strains, AD17, RHPA, and THRO, at an MOI of 0.1 for 4 h. Infected CD4⫹T cells were then transduced with anti-HIV Env GPI-X5, the GPI-AB65 negative control, or a mock control. Supernatants were collected, and the amounts of HIV-1 were determined using p24 ELISA at 14 days postinfection. Histograms show means⫾standard deviations (n⫽3). Statistical comparisons were done by ANOVA (****,P⬍0.0001; NS, not significant). All results shown are representative of three independent experiments.
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[image:9.585.43.497.70.410.2]anti-HIV Env intracellular antibody (46), they represent a distinct type of molecule and
are not limited to interacting with Env within the cell but also act to potently neutralize
viral entry (28). Thus, anti-HIV Env GPI-scFvs have a broader capacity to interfere with
different steps of the viral replication cycle than intracellular antibodies, which are not
expressed at the surface of cells and do not block viral entry.
Ultimately, we observed that the decrease in infectivity of the virions produced in
FIG 9Production and infectivity of HIV-1 induced from latency are inhibited by anti-HIV Env GPI-X5. (A) Expression levels of GPI-scFv constructs after treatment with TNF-␣. (B) Quantification of HIV-1 in cell supernatants by HIV p24 ELISA. (C) Relative infectivity of HIV-1 produced in the presence or absence of anti-HIV Env GPI-X5 or the GPI-AB65 negative control. The ACH-2 cell line was transduced with GPI-scFvs and treated with TNF-␣to induce production of HIV-1 LAI. Cells were harvested, and GPI-positive cells were quantified by staining for the His-tagged hinge region and analyzed by flow cytometry. Equal HIV p24 was used to infect TZM-bl cells. Histograms show means⫾standard deviations (n⫽3). Statistical comparisons were done by ANOVA (*,P⬍0.05;**,P⬍0.01,****,P⬍0.0001; NS, not significant). All results shown are representative of three independent experiments.
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[image:10.585.43.461.72.576.2]phosphatidylinositol-specific phospholipase C (PI-PLC) treatment gave inconclusive
results (data not shown). This result could be due to the fact that some cellular
GPI-anchored proteins incorporated into virions enhance infectivity by either
protect-ing the virus from complement (CD55 and CD59) or possibly promotprotect-ing interactions
with target cells (LFA-3) (47–49). Additionally, if anti-HIV Env GPI-scFvs were inhibiting
viral infectivity by binding Env gp120 on virions, we might not have observed the
decrease in Env gp120 in virions released from cells expressing GPI-X5 or GPI-PG16.
We also observed inhibition of virus release when CD4
⫹T cells were infected with
transmitter/founder HIV-1 strains followed by transduction with GPI-X5. GPI-X5 was
able to completely inhibit virus production 2 weeks after infection. GPI-X5 also inhibited
virus release and reduced the infectivity of progeny virions in a latent-infection model
using ACH-2 cells. These data show the efficacy of GPI-X5 even in infected primary
CD4
⫹T cells and a CD4
⫹T cell line.
Several gene therapy strategies have been developed over the years in order to
protect CD4
⫹T cells from HIV-1 infection. Creating a pool of virus-resistant cells by
targeting the receptors for HIV-1 CCR5 (50) and CXCR4 (51, 52) via a number of
RNA-based approaches such as ribozymes, short hairpin RNA (shRNA), and RNA
inter-ference (RNAi) (53–55), or by introducing mutations with zinc finger nucleases (56–58)
or CRISPR/Cas9 (59) has been somewhat successful. However, targeting one of the
coreceptors at a time could result in selection of HIV-1 for the other coreceptor. On the
other hand, deletion of CXCR4 is not ideal, especially in hematopoietic stem cells, as it
is an essential gene for development (60). However, with the use of the anti-HIV-1 Env
GPI-scFvs alone or in combination, we can block entry as well as other stages of the
HIV-1 life cycle, limiting the generation of escape mutants. Alternatively, lentiviral
vectors have been used to induce suicide genes (61, 62) or anti-HIV-1 genes (63, 64) in
T cells upon infection with HIV-1. Both of these strategies resulted in a selection
disadvantage of the T cell transduced with the vector or a loss in the functionality of
the T cells. Our preliminary studies with expressing the GPI-X5 on HIV-specific T cells
have not shown adverse effects on functionality. Quite interestingly, GPI-X5 may
enhance HIV-specific T cell clearance of infected cells (65). Potentially, the anti-HIV-1
GPI-scFvs could provide a new shield to protect CD4
⫹T cells from infection and
improve the immune status of 1-infected individuals, either by modifying
HIV-specific T cells for immunotherapy or modifying hematopoietic stem cells for
repopu-lating the infected host.
In summary, our study demonstrates the potential of the anti-HIV Env GPI-scFvs as
unique antiviral molecules which derive potent inhibitory activity against HIV-1
repli-cation by interfering with both early (receptor binding) and late (Env processing and
incorporation into virions) stages of the viral life cycle. A recent study by Ye et al. in
SCID-PBL mice infected with HIV demonstrated greater persistence of GPI-X5-modified
CD4
⫹T cells, further suggesting the potential use of anti-HIV Env GPI-scFvs as a genetic
intervention to protect CD4
⫹T cells from infection (35). It may also be important to
extend these studies to an immunocompetent host infection model such as the
simian-human immunodeficiency virus (SHIV)-rhesus macaque model in order to
eval-uate the capacity of anti-HIV Env GPI-scFv to protect permissive cells and restore
immunological control of the infection.
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MATERIALS AND METHODS
Viruses and cell lines.Proviral plasmids pNLAD8 (66), pNL4-3 (67), pRHPA, pTHRO (68–72), and pAD17 (73) were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP) (Germantown, MD). SIVmne027 was cloned and characterized as we previously described (74). 293T cells were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) (i.e., high-glucose DMEM supplemented with 10% fetal bovine serum [FBS], 2 mML-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100g/ml streptomycin). TZM-bl cells (75, 76) and ACH-2 cells (36, 37) were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP; Germantown, MD) and maintained in complete RPMI medium (i.e., high-glucose RPMI medium supplemented with 10% FBS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100g/ml streptomycin).
GPI-scFv constructs.The lentiviral vectors with scFv/IgG3 hinge/His tag/DAF (GPI-scFvs) minigenes incorporating the scFvs from AB65, X5, and PG16 were constructed as described previously (28). The GPI-scFv minigene cassettes were subcloned into pcDNA3 to generate AB65, pcDNA3-GPI-X5, and pcDNA-GPI-PG16.
HIV-1 transfection, infectivity assay, and Gag p24 ELISA.To examine the effect of GPI-scFvs on viral production and infectivity, 1g of HIV-1 plasmid NL4-3, AD8, and transmitter/founder HIV-1 strains RHPA, THRO, and AD17 and 1g of either pcDNA3-GPI-PG16, pcDNA3-GPI-X5, pcDNA3-GPI-AB65, or pcDNA3 DNA were cotransfected into 293T cells using X-tremeGENE 9 DNA transfection reagent according to the manufacturer’s protocol (Roche). After 48 h, supernatants were collected from trans-fected cultures of cells and passed through 0.45-m-pore-size syringe filters. HIV-1 p24gagantigen in the
supernatants was quantified by ELISA (Advanced Biosciences Laboratories). To compare the infectivity levels of the virus produced from the cotransfected cells, 1⫻104TZM-bl cells in DMEM complete with
30g/ml DEAE-dextran were added to wells of a 96-well plate, and triplicate cultures were incubated with supernatants from the cotransfected cells containing 0.25 ng of p24gagantigen. After 48 h, the
TZM-bl cells were washed once with PBS and lysed in 100l of Glo-Lysis buffer. Luciferase activity in 50
l of cell suspensions was measured by a BrightGlo luciferase assay with a luminometer according to the manufacturer’s instructions (Promega).
CD4ⴙT cell purification and infection.Human peripheral blood mononuclear cells (PBMCs) were
obtained from anonymous healthy donors through the Gulf Coast Blood Center (Houston, TX). Human primary CD4⫹T cells were enriched from PBMCs by negatively selecting magnetic beads according to the manufacturer’s instructions (Thermo Fisher Scientific) and resuspended in the complete RPMI 1640 medium (i.e., RPMI 1640 medium supplemented with 15% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100g/ml streptomycin) supplemented with human recombinant interleukin-2 ([rIL-2] 100 IU/ml; R&D Systems). Cells were then activated by costimulation using plates coated with anti-CD3/anti-CD28 monoclonal antibody (BD Pharmingen) for 3 days. Activation of cells was confirmed by fluorescence-assisted cell sorting (FACS) analysis for CD69 (BD Pharmingen). Activated CD4⫹T cells were infected with HIV-1 at a multiplicity of infection (MOI) of 0.1, spinoculated for 1 h at 4°C, and incubated at 37°C for 4 h, with shaking every 30 min. After infection the cells were washed with PBS twice and transduced with viral vectors expressing GPI-scFv.
Induction of HIV-1 from latently infected cells.ACH-2 cells were cultured in RPMI 1640 medium containing penicillin-streptomycin, glutamine, 10 mM HEPES, and 10% fetal bovine serum at 37°C in 5% CO2in the presence or absence of TNF-␣. ACH-2 cells were transduced with GPI-scFv before treatment
with TNF-␣.
FACS analysis.To study cell surface expression of scFv/hinge/His tag/DAF, 1⫻106mock-transfected
and scFv (AB65, X5, and PG16)/hinge/His tag/DAF-transfected 293T cells were incubated with a mouse anti-His tag antibody (Sigma) for 1 h on ice. Cells then were washed twice with FACS buffer (PBS containing 10% FBS and 0.1% azide) and stained with phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody (Sigma) for another hour on ice. Cells then were washed twice with FACS buffer and fixed with 1% formaldehyde in 0.5 ml of FACS buffer. Cells were analyzed with a Beckman-Coulter Gallios flow cytometer and Kaluza software.
Cell lysis and immunoblotting.Cell extracts were prepared by lysis of cells in ice-cold radioim-munoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) for immunoblotting containing protease inhibitors (Roche). Viral particles were concentrated from supernatants and lysed as we previously described (77). Proteins from virions or cells or immunoprecipitates were separated by SDS-PAGE using (4 to 15%) Tris-HCl Ready Gels (Bio-Rad) and transferred to nitrocellulose membranes for Western blotting. Blots were probed overnight at 4°C with antibodies to the HIV-1 Env protein, GP160B (HT3) (78, 79), from the NIH AIDS Research and Reference Reagent Program (ARRRP, Germantown, MD), HIV-1 p24gag(80), mouse
anti-His tag antibody (Sigma), or anti-actin (Sigma). Membranes were developed with Pierce ECL Western blotting chemiluminescence substrate for detection of horseradish peroxidase (HRP) (Thermo Scientific), and signals of bound antibodies were visualized by autoradiography. Densitom-etry was used to determine the relative level of each protein. The relative densities of bands were quantified using ImageJ according to the manual (81).
Statistical analyses.GraphPad Prism, version 6, software was used for graphing and statistically analyzing the data. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons was used to determine significant differences between three or more groups.Pvalues of⬍0.05 denote signifi-cance.
Misra et al. Journal of Virology
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This work was supported by research grants from the Chinese National Science
Foundation (31170871) and the National Science and Technology Major Project
(2014ZX10001-001) to P.Z., by a Chinese National Science Foundation-National
Insti-tutes of Health joint grant (81361120406-R01 AI106574) to P.Z. and J.T.K. and R21
AI116167 to J.T.K., and in part by the Baylor-UT Houston Center for AIDS Research
(AI036211).
REFERENCES
1. Monel B, Beaumont E, Vendrame D, Schwartz O, Brand D, Mammano F. 2012. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through Env-mediated fusion pores. J Virol 86:3924 –3933.https://doi.org/10.1128/JVI.06478-11. 2. Carr JM, Hocking H, Li P, Burrell CJ. 1999. Rapid and efficient cell-to-cell
transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Vi-rology 265:319 –329.https://doi.org/10.1006/viro.1999.0047.
3. Chen P, Hubner W, Spinelli MA, Chen BK. 2007. Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses. J Virol 81:12582–12595.https://doi.org/10.1128/JVI.00381-07.
4. Dimitrov DS, Willey RL, Sato H, Chang LJ, Blumenthal R, Martin MA. 1993. Quantitation of human immunodeficiency virus type 1 infection kinetics. J Virol 67:2182–2190.
5. Sourisseau M, Sol-Foulon N, Porrot F, Blanchet F, Schwartz O. 2007. Inefficient human immunodeficiency virus replication in mobile lympho-cytes. J Virol 81:1000 –1012.https://doi.org/10.1128/JVI.01629-06. 6. Hübner W, McNerney GP, Chen P, Dale BM, Gordon RE, Chuang FYS, Li
X-D, Asmuth DM, Huser T, Chen BK. 2009. Quantitative 3D video micros-copy of HIV transfer across T cell virological synapses. Science 323: 1743–1747.https://doi.org/10.1126/science.1167525.
7. McDonald D. 2003. Recruitment of HIV and Its receptors to dendritic cell-T cell junctions. Science 300:1295–1297.https://doi.org/10.1126/ science.1084238.
8. Sattentau Q. 2008. Avoiding the void: cell-to-cell spread of human viruses. Nat Rev Microbiol 6:815– 826.https://doi.org/10.1038/nrmicro1972. 9. Malbec M, Porrot F, Rua R, Horwitz J, Klein F, Halper-Stromberg A, Scheid
JF, Eden C, Mouquet H, Nussenzweig MC, Schwartz O. 2013. Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission. J Exp Med 210:2813–2821.https://doi.org/10.1084/jem.20131244.
10. Abela IA, Berlinger L, Schanz M, Reynell L, Günthard HF, Rusert P, Trkola A. 2012. Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies. PLoS Pathog 8:e1002634.https://doi .org/10.1371/journal.ppat.1002634.
11. Iwami S, Takeuchi JS, Nakaoka S, Mammano F, Clavel F, Inaba H, Ko-bayashi T, Misawa N, Aihara K, Koyanagi Y, Sato K, Adachi A, Gendelman H, Koenig S, Folks T, Willey R, Rabson A, Martin M, Agosto L, Zhong P, Munro J, Mothes W, Carr J, Hocking H, Li P, Burrell C, Chen P, Hübner W, Spinelli M, Chen B, Dimitrov D, Willey R, Sato H, Chang L, Blumenthal R, Martin M, Fukuhara M, Iwami S, Sato K, Nishimura Y, Shimizu H, Aihara K, Koyanagi Y, Inaba H, Iwami S, Holder B, Beauchemin C, Morita S, Tada T, Sato K, Igarashi T, et al. 2015. Cell-to-cell infection by HIV contributes over half of virus infection. Elife 4:284 –291.https://doi.org/10.7554/eLife .08150.
12. Murooka TT, Deruaz M, Marangoni F, Vrbanac VD, Seung E, von Andrian UH, Tager AM, Luster AD, Mempel TR. 2012. HIV-infected T cells are
migratory vehicles for viral dissemination. Nature 490:283–287.https:// doi.org/10.1038/nature11398.
13. Sigal A, Kim JT, Balazs AB, Dekel E, Mayo A, Milo R, Baltimore D. 2011. Cell-to-cell spread of HIV permits ongoing replication despite antiretro-viral therapy. Nature 477:95–98.https://doi.org/10.1038/nature10347. 14. Su B, Xu K, Lederle A, Peressin M, Biedma ME, Laumond G, Schmidt S,
Decoville T, Proust A, Lambotin M, Holl V, Moog C. 2012. Neutralizing antibodies inhibit HIV-1 transfer from primary dendritic cells to autolo-gous CD4 T lymphocytes. Blood 120:3708 –3717. https://doi.org/10 .1182/blood-2012-03-418913.
15. Sagar M, Akiyama H, Etemad B, Ramirez N, Freitas I, Gummuluru S. 2012. Transmembrane domain membrane proximal external region but not surface unit-directed broadly neutralizing HIV-1 antibodies can restrict dendritic cell-mediated HIV-1 trans-infection. J Infect Dis 205: 1248 –1257.https://doi.org/10.1093/infdis/jis183.
16. Massanella M, Puigdomènech I, Cabrera C, Fernandez-Figueras MT, Aucher A, Gaibelet G, Hudrisier D, García E, Bofill M, Clotet B, Blanco J. 2009. Antigp41 antibodies fail to block early events of virological syn-apses but inhibit HIV spread between T cells. AIDS 23:183–188.https:// doi.org/10.1097/QAD.0b013e32831ef1a3.
17. Martin N, Welsch S, Jolly C, Briggs JAG, Vaux D, Sattentau QJ. 2010. Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition. J Virol 84: 3516 –3527.https://doi.org/10.1128/JVI.02651-09.
18. van Montfort T, Nabatov AA, Geijtenbeek TBH, Pollakis G, Paxton WA. 2007. Efficient capture of antibody neutralized HIV-1 by cells expressing DC-SIGN and transfer to CD4⫹ T lymphocytes. J Immunol 178: 3177–3185.https://doi.org/10.4049/jimmunol.178.5.3177.
19. van Montfort T, Thomas AAM, Pollakis G, Paxton WA. 2008. Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4⫹T lymphocytes in trans. J Virol 82:7886 –7896.
https://doi.org/10.1128/JVI.00245-08.
20. Reh L, Magnus C, Schanz M, Weber J, Uhr T, Rusert P, Trkola A. 2015. Capacity of broadly neutralizing antibodies to inhibit HIV-1 cell-cell transmission is strain- and epitope-dependent. PLoS Pathog 11: e1004966.https://doi.org/10.1371/journal.ppat.1004966.
21. Chazal N, Gerlier D. 2003. Virus entry, assembly, budding, and mem-brane rafts. Microbiol Mol Biol Rev 67:226 –237.https://doi.org/10.1128/ MMBR.67.2.226-237.2003.
22. Liao Z, Graham DR, Hildreth JE. 2003. Lipid rafts and HIV pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cells. AIDS Res Hum Retroviruses 19:675– 687.https://doi .org/10.1089/088922203322280900.
23. Campbell S, Crowe S, Mak J. 2001. Lipid rafts and HIV-1: from viral entry to assembly of progeny virions. J Clin Virol 22:217–227.https://doi.org/ 10.1016/S1386-6532(01)00193-7.
24. Harder T, Engelhardt KR. 2004. Membrane domains in lymphocytes—
on November 6, 2019 by guest
http://jvi.asm.org/
from lipid rafts to protein scaffolds. Traffic 5:265–275.https://doi.org/10 .1111/j.1600-0854.2003.00163.x.
25. Popik W, Alce TM, Au W-C. 2002. Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4⫹T cells. J Virol 76:4709 – 4722.https://doi.org/10.1128/
JVI.76.10.4709-4722.2002.
26. Carter GC, Bernstone L, Sangani D, Bee JW, Harder T, James W. 2009. HIV entry in macrophages is dependent on intact lipid rafts. Virology 386: 192–202.https://doi.org/10.1016/j.virol.2008.12.031.
27. Jolly C, Sattentau QJ. 2005. Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity. J Virol 79:12088 –12094.https://doi.org/10.1128/JVI.79.18.12088-12094.2005. 28. Wen M, Arora R, Wang H, Liu L, Kimata JT, Zhou P. 2010. GPI-anchored
single chain Fv—an effective way to capture transiently-exposed neu-tralization epitopes on HIV-1 envelope spike. Retrovirology 7:79.https:// doi.org/10.1186/1742-4690-7-79.
29. Liu L, Wang W, Yang L, Ren H, Kimata JT, Zhou P. 2013. Trimeric glycosylphosphatidylinositol-anchored HCDR3 of broadly neutralizing antibody PG16 is a potent HIV-1 entry inhibitor. J Virol 87:1899 –1905.
https://doi.org/10.1128/JVI.01038-12.
30. Liu L, Wang W, Matz J, Ye C, Bracq L, Delon J, Kimata JT, Chen Z, Benichou S, Zhou P. 2016. The glycosylphosphatidylinositol-anchored variable region of llama heavy chain-only antibody JM4 efficiently blocks both cell-free and T cell-T cell transmission of human immu-nodeficiency virus type 1. J Virol 90:10642–10659.https://doi.org/10 .1128/JVI.01559-16.
31. Liu L, Wen M, Zhu Q, Kimata JT, Zhou P. 2016. Glycosyl phosphatidylinositol-anchored C34 peptide derived from human immunodeficiency virus type 1 Gp41 is a potent entry inhibitor. J Neuroimmune Pharmacol 11:601– 610.
https://doi.org/10.1007/s11481-016-9681-x.
32. Medof ME, Kinoshita T, Nussenzweig V. 1984. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med 160: 1558 –1578.https://doi.org/10.1084/jem.160.5.1558.
33. Liu L, Wen M, Wang W, Wang S, Yang L, Liu Y, Qian M, Zhang L, Shao Y, Kimata JT, Zhou P. 2011. Potent and broad anti-HIV-1 activity exhibited by a glycosyl-phosphatidylinositol-anchored peptide derived from the CDR H3 of broadly neutralizing antibody PG16. J Virol 85:8467– 8476.
https://doi.org/10.1128/JVI.00520-11.
34. Moulard M, Phogat SK, Shu Y, Labrijn AF, Xiao X, Binley JM, Zhang M-Y, Sidorov IA, Broder CC, Robinson J, Parren PWHI, Burton DR, Dimitrov DS. 2002. Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4-CCR5 complexes. Proc Natl Acad Sci U S A 99:6913– 6918.https://doi.org/10.1073/pnas.102562599. 35. Ye C, Wang W, Cheng L, Li G, Wen M, Wang Q, Zhang Q, Li D, Zhou P,
Su L. 2017. Glycosylphosphatidylinositol-anchored anti-HIV scFv effi-ciently protects CD4 T cells from HIV-1 infection and deletion in hu-PBL mice. J Virol 91:e01389-16.https://doi.org/10.1128/JVI.01389-16. 36. Folks TM, Clouse KA, Justement J, Rabson A, Duh E, Kehrl JH, Fauci AS.
1989. Tumor necrosis factor alpha induces expression of human immu-nodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A 86:2365–2368.https://doi.org/10.1073/pnas.86.7.2365. 37. Clouse KA, Powell D, Washington I, Poli G, Strebel K, Farrar W, Barstad P,
Kovacs J, Fauci AS, Folks TM. 1989. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 142:431– 438.
38. Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG. 1998. A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum. J Cell Biol 140:525–540.
https://doi.org/10.1083/jcb.140.3.525.
39. Malagolini N, Cavallone D, Serafini-Cessi F. 1997. Intracellular transport, cell-surface exposure and release of recombinant Tamm-Horsfall glyco-protein. Kidney Int 52:1340 –1350.https://doi.org/10.1038/ki.1997.459. 40. Zhang MY, Shu Y, Rudolph D, Prabakaran P, Labrijn AF, Zwick MB, Lal RB,
Dimitrov DS. 2004. Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J Mol Biol 335:209 –219.https://doi.org/10 .1016/j.jmb.2003.09.055.
41. Thali M, Moore JP, Furman C, Charles M, Ho DD, Robinson J, Sodroski J. 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol 67:3978 –3988.
42. Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, Franti M, Binley J, Vivona V, Grundner C, Huang C-C, Venturi M, Petropoulos CJ, Wrin T,
Dimitrov DS, Robinson J, Kwong PD, Wyatt RT, Sodroski J, Burton DR. 2003. Access of antibody molecules to the conserved coreceptor bind-ing site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557–10565.https://doi.org/ 10.1128/JVI.77.19.10557-10565.2003.
43. Darbha R, Phogat S, Labrijn AF, Shu Y, Gu Y, Andrykovitch M, Zhang M-Y, Pantophlet R, Martin L, Vita C, Burton DR, Dimitrov DS, Ji X. 2004. Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope. Biochemistry 43:1410 –1417.https://doi.org/10 .1021/bi035323x.
44. Crooks ET, Moore PL, Richman D, Robinson J, Crooks JA, Franti M, Schülke N, Binley JM. 2005. Characterizing anti-HIV monoclonal antibod-ies and immune sera by defining the mechanism of neutralization. Hum Antibodies 14:101–113.
45. Ali A, Kitchen SG, Chen IS, Ng HL, Zack JA, Yang OO. 2016. HIV-1-specific chimeric antigen receptors based on broadly neutralizing antibodies. J Virol 90:6999 –7006.https://doi.org/10.1128/JVI.00805-16.
46. Zhou P, Goldstein S, Devadas K, Tewari D, Notkins AL. 1998. Cells transfected with a non-neutralizing antibody gene are resistant to HIV infection: targeting the endoplasmic reticulum and trans-Golgi network. J Immunol 160:1489 –1496.
47. Bastiani L, Laal S, Kim M, Zolla-Pazner S. 1997. Host cell-dependent alterations in envelope components of human immunodeficiency virus type 1 virions. J Virol 71:3444 –3450.
48. Saifuddin M, Hedayati T, Atkinson JP, Holguin MH, Parker CJ, Spear GT. 1997. Human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46 at levels that protect from complement-mediated destruction. J Gen Virol 78:1907–1911.https://doi.org/10.1099/0022-1317-78-8-1907. 49. Saifuddin M, Parker CJ, Peeples ME, Gorny MK, Zolla-Pazner S, Ghassemi
M, Rooney IA, Atkinson JP, Spear GT. 1995. Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in comple-ment resistance of cell line-derived and primary isolates of HIV-1. J Exp Med 182:501–509.https://doi.org/10.1084/jem.182.2.501.
50. Feng Y, Leavitt M, Tritz R, Duarte E, Kang D, Mamounas M, Gilles P, Wong-Staal F, Kennedy S, Merson J, Yu M, Barber JR. 2000. Inhibition of CCR5-dependent HIV-1 infection by hairpin ribozyme gene therapy against CC-chemokine receptor 5. Virology 276:271–278.https://doi.org/ 10.1006/viro.2000.0536.
51. Bai X, Chen JD, Yang AG, Torti F, Chen SY. 1998. Genetic co-inactivation of macrophage- and T-tropic HIV-1 chemokine coreceptors CCR-5 and CXCR-4 by intrakines. Gene Ther 5:984 –994.https://doi.org/10.1038/sj .gt.3300667.
52. BouHamdan M, Strayer DS, Wei D, Mukhtar M, Duan LX, Hoxie J, Pomer-antz RJ. 2001. Inhibition of HIV-1 infection by down-regulation of the CXCR4 co-receptor using an intracellular single chain variable fragment against CXCR4. Gene Ther 8:408 – 418. https://doi.org/10.1038/sj.gt .3301411.
53. Anderson J, Li M-J, Palmer B, Remling L, Li S, Yam P, Yee J-K, Rossi J, Zaia J, Akkina R. 2007. Safety and efficacy of a lentiviral vector containing three anti-HIV genes—CCR5 ribozyme, tat-rev siRNA, and TAR decoy—in SCID-hu mouse-derived T cells. Mol Ther 15:1182–1188.https://doi.org/ 10.1038/sj.mt.6300157.
54. Anderson JS, Javien J, Nolta JA, Bauer G. 2009. Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5␣ protein, a CCR5 shRNA, and a TAR decoy. Mol Ther 17: 2103–2114.https://doi.org/10.1038/mt.2009.187.
55. Kim S-S, Peer D, Kumar P, Subramanya S, Wu H, Asthana D, Habiro K, Yang Y-G, Manjunath N, Shimaoka M, Shankar P. 2010. RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 18:370 –376.https://doi.org/10.1038/mt.2009.271. 56. Wilen CB, Wang J, Tilton JC, Miller JC, Kim KA, Rebar EJ, Sherrill-Mix SA, Patro SC, Secreto AJ, Jordan APO, Lee G, Kahn J, Aye PP, Bunnell BA, Lackner AA, Hoxie JA, Danet-Desnoyers GA, Bushman FD, Riley JL, Gregory PD, June CH, Holmes MC, Doms RW. 2011. Engineering HIV-resistant human CD4⫹T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog 7:e1002020.https://doi.org/10.1371/journal.ppat.1002020. 57. Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, Cannon PM. 2010. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 28:839 – 847.https://doi.org/ 10.1038/nbt.1663.
58. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee Y-L, Guschin DY, Rupniewski I, Waite AJ, Carpenito C,
Misra et al. Journal of Virology
on November 6, 2019 by guest
http://jvi.asm.org/
tion against HIV type 1 infection in the human promonocytic cell line U937. Hum Gene Ther 6:1437–1445.https://doi.org/10.1089/hum.1995 .6.11-1437.
62. Mautino MR, Morgan RA. 2002. Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes. AIDS Patient Care STDS 16:11–26.https://doi.org/10.1089/108729102753429361.
63. Li M-J, Bauer G, Michienzi A, Yee J-K, Lee N-S, Kim J, Li S, Castanotto D, Zaia J, Rossi JJ. 2003. Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol Ther 8:196 –206.https:// doi.org/10.1016/S1525-0016(03)00165-5.
64. Michienzi A, Castanotto D, Lee N, Li S, Zaia JA, Rossi JJ. 2003. RNA-mediated inhibition of HIV in a gene therapy setting. Ann N Y Acad Sci 1002:63–71.https://doi.org/10.1196/annals.1281.008.
65. Patel S, Wright KE, Misra A, Zhou P, Kimata JT, Bollard C, Cruz CR. 2017. HIV-specific T cells expressing an X5-GPI artificial receptor can suppress HIV replication in vitro—implications for a cure strategy for HIV⫹ indi-viduals with hematologic malignancies. Cytotherapy 19(Suppl):S7–S8.
https://doi.org/10.1016/j.jcyt.2017.02.005.
66. Freed EO, Englund G, Martin MA. 1995. Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J Virol 69:3949 –3954.
67. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284 –291.
68. Keele BF, Tazi L, Gartner S, Liu Y, Burgon TB, Estes JD, Thacker TC, Crandall KA, McArthur JC, Burton GF. 2008. Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1. J Virol 82:5548 –5561.https://doi.org/10.1128/JVI.00124-08. 69. Salazar-Gonzalez JF, Bailes E, Pham KT, Salazar MG, Guffey MB, Keele BF,
Derdeyn CA, Farmer P, Hunter E, Allen S, Manigart O, Mulenga J, Anderson JA, Swanstrom R, Haynes BF, Athreya GS, Korber BTM, Sharp PM, Shaw GM, Hahn BH. 2008. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J Virol 82:3952–3970.https://doi .org/10.1128/JVI.02660-07.
70. Salazar-Gonzalez JF, Salazar MG, Keele BF, Learn GH, Giorgi EE, Li H, Decker JM, Wang S, Baalwa J, Kraus MH, Parrish NF, Shaw KS, Guffey MB, Bar KJ, Davis KL, Ochsenbauer-Jambor C, Kappes JC, Saag MS, Cohen MS, Mulenga J, Derdeyn CA, Allen S, Hunter E, Markowitz M, Hraber P, Perelson AS, Bhattacharya T, Haynes BF, Korber BT, Hahn BH, Shaw GM.
73. Parrish NF, Gao F, Li H, Giorgi EE, Barbian HJ, Parrish EH, Zajic L, Iyer SS, Decker JM, Kumar A, Hora B, Berg A, Cai F, Hopper J, Denny TN, Ding H, Ochsenbauer C, Kappes JC, Galimidi RP, West AP, Bjorkman PJ, Wilen CB, Doms RW, O’Brien M, Bhardwaj N, Borrow P, Haynes BF, Muldoon M, Theiler JP, Korber B, Shaw GM, Hahn BH. 2013. Phenotypic properties of transmitted founder HIV-1. Proc Natl Acad Sci U S A 110:6626 – 6633.
https://doi.org/10.1073/pnas.1304288110.
74. Kimata JT, Mozaffarian A, Overbaugh J. 1998. A lymph node-derived cytopathic simian immunodeficiency virus Mne variant replicates in nonstimulated peripheral blood mononuclear cells. J Virol 72:245–256. 75. Derdeyn CA, Decker JM, Sfakianos JN, Wu X, O’Brien WA, Ratner L, Kappes JC, Shaw GM, Hunter E. 2000. Sensitivity of human immunode-ficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 74: 8358 – 8367.https://doi.org/10.1128/JVI.74.18.8358-8367.2000. 76. Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu X,
Shaw GM, Kappes JC. 2002. Emergence of resistant human immunode-ficiency virus type 1 in patients receiving fusion inhibitor (T-20) mono-therapy. Antimicrob Agents Chemother 46:1896 –1905.https://doi.org/ 10.1128/AAC.46.6.1896-1905.2002.
77. Arora R, Bull L, Siwak EB, Thippeshappa R, Arduino RC, Kimata JT. 2010. Dendritic cell-mediated HIV-1 infection of T cells demonstrates a direct relationship to plasma viral RNA levels. J Acquir Immune Defic Syndr 54:115–121.
78. Matsushita S, Robert-Guroff M, Rusche J, Koito A, Hattori T, Hoshino H, Javaherian K, Takatsuki K, Putney S. 1988. Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J Virol 62:2107–2114.
79. Rusche JR, Lynn DL, Robert-Guroff M, Langlois AJ, Lyerly HK, Carson H, Krohn K, Ranki A, Gallo RC, Bolognesi DP. 1987. Humoral immune response to the entire human immunodeficiency virus envelope glyco-protein made in insect cells. Proc Natl Acad Sci U S A 84:6924 – 6928.
https://doi.org/10.1073/pnas.84.19.6924.
80. Chesebro B, Wehrly K, Nishio J, Perryman S. 1992. Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J Virol 66:6547– 6554.
81. Ferreira T, Rasband W. 2012. ImageJ user guide. National Institutes of Health, Bethesda, MD.
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