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Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity

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Anisha Misra,

a

Emile Gleeson,

a

Weiming Wang,

b

Chaobaihui Ye,

b

Paul Zhou,

b

Jason T. Kimata

a

aDepartment 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

gag

was

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

gag

was 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

gag

production 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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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

gag

were 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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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

gag

production 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.

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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

gag

was 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

gag

was 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

gag

were 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.

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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

T 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

gag

was 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.

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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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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 CD4T 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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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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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, 100␮g/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, 100␮g/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, 1␮g of HIV-1 plasmid NL4-3, AD8, and transmitter/founder HIV-1 strains RHPA, THRO, and AD17 and 1␮g 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

30␮g/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 100␮l 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).

CD4T 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 100␮g/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.

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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).

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Figure

FIG 1 Expression levels of GPI-scFv constructs after cotransfection. 293T cells were transfected withGPI-scFv constructs and harvested, and GPI-positive cells were quantified by staining for the His-taggedhinge region by flow cytometry
FIG 2 Production of virions is significantly decreased by anti-HIV Env GPI-scFvs. 293T cells were cotransfected with
FIG 4 Production and infectivity of SIV is unaffected by anti-HIV Env GPI-scFvs. (A) Quantification of SIVproduced in the presence or absence of GPI-scFvs using an SIV p27 ELISA
FIG 5 Processing of Env gp160 precursors in cells is inhibited by anti-HIV Env GPI-scFvs
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