www.elsevier.com/locate/yviro

Virology 324 (2004) 60–66

Herpesvirus saimiri-encoded proteins Tip and StpC modulate human

immunodeficiency virus type 1 replication in T-cell lines and

lymphocytes independently of viral tropism

Andrea D. Raymond,a Muneer G. Hasham,a

Alexander Y. Tsygankov,a and Earl E. Hendersona,b,c,*

aDepartment of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USAbCenter for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA 19140, USA

cThe Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA

Received 14 November 2003; returned to author for revision 9 January 2004; accepted 19 March 2004

Abstract

Herpesvirus saimiri (HVS)-transformed T-lymphocytes are permissive for both X4 and R5 strains of human immunodeficiency virus type

1 (HIV-1). HVS-encoded proteins tyrosine-kinase interacting protein (Tip) and saimiri transformation-associated protein subgroup C (StpC)

were previously implicated in altering HIV permissiveness. MOLT4 cells expressing StpC or StpC and Tip are permissive for X4 strains of

HIV-1. In contrast, HIV-1 was restricted in MOLT4 cells expressing Tip alone. Here we show that MOLT4 cells and primary lymphocytes

expressing StpC are permissive for R5 strains of HIV-1 while Tip expression restricted R5 strains. These results suggest that intracellular

immunization with Tip and StpC could be developed as models for therapeutic strategies targeting both X4 and R5 strains of HIV-1.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Herpesvirus saimiri; HIV-1; Viral tropism; T-cell

Introduction

Human peripheral blood T-lymphocytes have been suc-

cessfully transformed in vitro by infection with Herpesvirus

saimiri (HVS) subgroup C strains (Biesinger et al., 1992).

HVS-transformed T-cell lines retain properties of normal T-

cells including antigen specificity and a mature activated

phenotype (Broker et al., 1993). As a result, HVS-trans-

formed T-cells have been used as a model to study T-cell

biology (Meinl and Hohlfeld, 2000). In that regard, HVS-

transformed T-cells are highly permissive for both T-tropic

(X4) (Nick et al., 1993; Saha et al., 1997; Vella et al., 1997)

and M-tropic (R5) strains of human immunodeficiency virus

type 1 (HIV-1) (Vella et al., 1999). HVS-immortalized human

lymphocytes have been utilized for the isolation, propaga-

tion, and analysis of diverse strains of HIV-1 (Vella et al.,

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2004.03.021

* Corresponding author. Department of Microbiology and Immunology,

Temple University School of Medicine, 3400 North Broad Street,

Philadelphia, PA 19140. Fax: +1-215-707-7788.

E-mail address: eehender@temple.edu (E.E. Henderson).

2002). Evidence has accumulated that saimiri transformation-

associated protein subgroup C (StpC) and tyrosine-kinase

interacting protein (Tip), two unique proteins of subgroup C,

are responsible for the permissive phenotype of HVS-trans-

formed T-cells. Tip and StpC are required for HVS-induced

transformation and are translated from a bicistronic RNA

message (reviewed in Tsygankov and Romano, 1999).

MOLT4 cells retrovirally transduced to express StpC are

permissive for X4 strains of HIV-1, whereas Tip expression in

MOLT4 dramatically restricted the replication of X4 strains

of HIV-1 (Henderson et al., 1999). In double transductants,

the StpC phenotype was dominant and thus consistent with

the observation that HVS-transformed T-cells are permissive

for HIV-1. The ability of Tip and StpC to modulate the

replication of X4 strains of HIV-1 suggested that therapeutic

strategies targeting HIV-1 could be modeled on the ectopic

expression of Tip or StpC, or both. However, therapeutic

strategies targeting HIV-1 would need to inhibit both X4 and

R5 strains of HIV-1 to be effective. For example, a strategy

designed to target HIV-1 based on inhibiting gp120-mediated

signal transduction in T-lymphocytes restricted replication of

A.D. Raymond et al. / Virology 324 (2004) 60–66 61

X4 but not R5 strains of HIV-1, limiting therapeutic useful-

ness (Popik and Pitha, 2000). In this report, we show that Tip

and StpCmodulate the replication of R5 as well as dual tropic

strains of HIV-1 in a manner similar to that observed for X4

strains of HIV-1, and could be developed as models for

therapeutic strategies. Therapeutics effecting both X4 and

R5 strains of HIV would be extremely advantageous, since

there is a direct correlation between the phenotypic switch

from R5 to X4 tropism and increased HIV-1 pathogenesis

(Glushakova et al., 1998).

Results

Tip and StpC affect replication of M-tropic (R5) and dual

tropic strains of HIV-1

MOLT4 transduced with murine stem cell virus

(MSCV)-based vectors encoding Tip and/or StpC (Merlo

et al., 1998) were used to study the effects of these

Fig. 1. Replication of R5 strains of HIV-1 (Ba-L and SF162) in MOLT4 cells trans

p24 by ELISA were measured 7 days following infection. PN = Pac/Neo; TN

transductants) are denoted as follows: C2 = StpC/Tip/PN clone C2; D2 = StpC/Tip

infected pac-, neo-, or Tip-transduced MOLT4 cells were used to infect PBMCs. V

measuring p24 antigen levels in culture supernatants using a capture ELISA. HIV-1

with supernatants from MOLT4 transduced with pac/neo or StpC-pac/neo then inf

supernatants was determined using p24 ELISA 10 days post infection. *P < 0.05

proteins on the replication of R5 strains of HIV-1. When

compared to control cells transduced with the empty

vectors (pac/neo), Tip-expressing cells exhibit approxi-

mately a 10-fold restriction in the replication of R5 strains

of HIV-1, as measured by syncytia formation and the

accumulation of p24 in culture supernatants (Fig. 1A).

Consistent with these results, HIV-1-induced cytopathic

effects (CPE) were ameliorated 10-fold and cell survival

enhanced 14- to 20-fold by Tip expression (Table 1). In

contrast, StpC-expressing cells exhibited a 3- to 6-fold

increase in p24 production and syncytia as well as greatly

increased CPE and reduced survival when compared to

MOLT4 cells transduced with empty vector alone (Fig.

1A; Table 1). In MOLT4 clones expressing both Tip and

StpC, the StpC phenotype dominated, consistent with the

observation that HVS-transformed T cells are highly per-

missive for R5 strains of HIV-1. We examined whether the

ability of Tip and StpC to affect HIV-1 infection in

transduced MOLT4 cells could be explained by changes

in the expression of HIV-1 receptor or coreceptors. Flow

duced to express Tip and/or StpC. (A) Syncytia (insert) and accumulation of

= Tip/Neo; SP = StpC/Pac. Clones expressing both StpC and Tip (dual

/PN clone D2; D3 = StpC/Tip/PN clone D3. (B) Culture supernatants from

iral replication in PBMCs was monitored following 10 days of infection by

strains were used: Ada-M and JRFL. *P < 0.05. (C) PBMCs were infected

ected with Ada-M or JRFL. Accumulation of p24 antigen in PBMC culture

determined by Student’s t test (n = 3).

Table 1

Colony formation by Molt4 cells transduced with StpC, Tip, or StpC and

Tip following infection with R5 stains of HIV-1

Cell lines Percentage colony-forming ability as measured by

limiting dilution:

Mock SF162 Bal

Single transductants

Molt4 Pac/Neo >90 5.0 2.5

Molt4 Tip,

Pac/Neo

>90 70 20

Molt4 StpC,

Pac/Neo

>90 0.001 V0.0001

Double transductants

Molt4 StpC/

Tip C2

>90 0.001 V0.0001

Molt4 StpC/

Tip D3

>90 0.001 0.001

A.D. Raymond et al. / Virology 324 (2004) 60–6662

cytometric analysis of MOLT4 cells expressing StpC or

Tip did not reveal significant differences in the mean

fluorescent intensity (MFI) or the percent of cells positive

for CD4, CCR5, or CXCR4 surface expression that would

explain the significant differences in susceptibility to HIV-

1 infection observed (data not shown).

Expression of R5 strains of HIV-1 in latently infected

MOLT4 clones expressing Tip or StpC

The survival of HIV-1-infected MOLT4 cells expressing

Tip is significantly increased more than that of MOLT4

Fig. 2. Tip and StpC expression in PBMCs modulates HIV infection. (A) Expressio

in PBMCs following transduction with lentiviral vectors driving the expression o

SupT1. * = Significantly different from cells transduced with matched control an

cells transduced with empty vector alone and surviving

colonies are easily established (Table 1). In contrast,

MOLT4 cells expressing StpC rarely survive infection

with R5 strains of HIV-1. However, it is possible to

establish cell lines from HIV-infected MOLT4 cells

expressing StpC, albeit at a low frequency (Table 1).

Clones established from Tip- and StpC-expressing MOLT4

cells, which survived HIV-1 infection, harbor HIV-1

proviral DNA (data not shown). To further assess the

virological parameters in these surviving clones, cell-free

supernatants were analyzed for infectious virus. Infectious

HIV-1 released by MOLT4 transduced to express Tip was

reduced 70.5–99.9% when compared to MOLT4 cells

transduced with empty vector (Fig. 1B). In contrast, there

was a 3- to 11-fold increase in infectious virus released

from surviving clones of MOLT4 transduced with StpC

when compared to surviving clones transduced with the

empty vectors (pac/neo) alone (Fig. 1C).

Transduction of peripheral blood mononuclear cells

(PBMCs) with Tip or StpC altered replication of both X4

and R5 strains of HIV-1

Using a lentiviral vector system, efficient expression of

both Tip and StpC was achieved in MOLT4 cells and

nonimmortalized lymphocytes (Hasham and Tsygankov,

2004). To determine whether expression of Tip and StpC

in lymphocytes affects HIV-1 replication, PBMCs were

transduced to express Tip or StpC (Fig. 2A) and then

n of Tip and StpC in PBMCs. (B) Infected-centers assay: HIV-1 replication

f Tip or StpC as measured by syncytia formation following co-culture with

tibiotic selection vectors. P < 0.05.

A.D. Raymond et al. / Virology 324 (2004) 60–66 63

infected with IIIB or SF162 strains of HIV-1. Tip expres-

sion inhibited virus-induced CPE 2-fold as measured by

syncytia induction, while StpC-expression induced a 10-

fold enhancement of syncytia induction by PBMCs (Fig.

2B). We did not observe phenotypic changes in uninfected

T-cells transduced with lentiviral vectors encoding StpC or

Tip.

Replication of HIV-1 is modulated in human umbilical cord

blood leukocytes (HUCLs) transduced with lentiviral

vectors encoding Tip or StpC

Potential therapeutic applications of Tip and/or StpC may

involve the use of progenitor cells derived from HUCLs. To

this end, HUCLs were transduced with lentiviral vectors

encoding Tip or StpC, placed on antibiotic selection, and

then enriched for either lymphocytes or monocytes by

culturing in medium containing IL-2 and M-CSF, respec-

tively. The enriched cells were then infected with either X4

or R5 strains of HIV-1. Replication of R5 strains of HIV-1

was quantitated in enriched monocytes and T-cells derived

from HUCLs transduced with lentiviral vectors encoding

Tip or StpC. R5 and X4 strains of HIV-1 were restricted in

cells derived from Tip-transduced HUCLs (Figs. 3A,B).

Cells derived from StpC-transduced HUCL were permissive

for R5 and X4 strains of HIV-1 (Figs. 3C,D). These results

show that expression of Tip and StpC can modulate repli-

cation of both X4 and R5 strains of HIV-1 in primary

lymphocytes.

Fig. 3. Effects of HVS-Tip and StpC on X4 and R5 HIV-1 replication in HUCL

supernatant of infected human umbilical cord blood-derived macrophage at da

lymphocytes (A, C) and HUCL-derived macrophage (B, D) were transduced with

then infected with RF or SF162. * = Statistical significance determined using Stu

Discussion

Tip and StpCmodulated replication of R5 strains of HIV-1

in MOLT4, PBMCs, and HUCL-derived cells in a manner

similar to that observed for X4 strains. Replication of R5

strains of HIV-1 in Tip-transduced MOLT4 and primary cells

was dramatically restricted, as measured by p24 antigen and

syncytia formation. Tip expression significantly increased

cell survival of HIV-1-infected cells and lowered both the

production of infectious virus and syncytia in surviving

clones. In contrast, replication of R5 strains of HIV-1 was

dramatically enhanced in MOLT4, PBMCs, and HUCL-

derived cells expressing StpC. The mechanism whereby Tip

and StpC mediate effects on HIV-1 replication is not known.

However, we posit that the dichotomous effects of Tip and

StpC on HIV-1 replication are due to differences in how these

HVS-encoded proteins affect signal transduction pathways

critical to the HIV-1 life cycle. Studies have shown that both

Tip and StpC interact with various components of T-cell

signaling pathways (reviewed in Tsygankov and Romano,

1999). StpC activates Ras and NF-nB signaling pathways

(Guo et al., 1998; Jung and Desrosiers, 1995). Both Ras-

activated AP-1 and NF-nB transcription factors have been

shown to up-regulate expression of the HIV-1 LTR (Rohr et

al., 2003). These findings are consistent with the effects of

StpC on HIV-1 replication.

Tip-mediated restriction of HIV-1 replication could be a

result of Tip interaction with Lck, a T-cell-specific protein

tyrosine kinase, which is critical for T-cell activation.

-derived monocytes and T-lymphocytes. Concentration of p24 released in

ys 14 and 21 was determined using p24 capture ELISA. HUCL-derived

Neo, Tip/Neo, Pac, or StpC/Pac vectors, placed on selection for 7 days, and

dent’s t test. P < 0.05.

A.D. Raymond et al. / Virolo64

Previous studies have demonstrated that Tip directly binds

and up-regulates Lck activity (Biesinger et al., 1995;

Wiese et al., 1996). However, a reduction in Lck activity

in Tip-transduced Jurkat T-cells has also been reported

(Guo et al., 1997). Tip-expressing MOLT4 cells used in

our study did not display significant alterations in Lck

activity but showed a dramatic increase in overall protein

tyrosine phosphorylation (Merlo et al., 1998).

Here, we report that replication of X4 and R5 strains of

HIV-1 are modulated by StpC and Tip in the same manner;

StpC enhanced, whereas Tip restricted viral replication. This

is advantageous, because the ectopic expression of these

proteins in immune cells could be developed as models for

therapeutic strategies against both M-and T-tropic strains of

HIV-1. For example, Tip-expressing T-cells would potential-

ly be protected from the cytopathic effects of HIV-1. StpC

expression vectors could be utilized to induce replication-

competent reservoirs sensitizing them to high activity anti-

retroviral therapy (HAART). Since wild-type and quasi-

species of HIV-1 R5 and X4 variants persist during pro-

longed antiretroviral drug therapy (van Rij et al., 2002), it is

imperative that novel therapies directed against HIV-1 be

effective against R5 and X4 strains. Here we demonstrate

that the ectopic expression of Tip and StpC can modulate the

replication of R5 and X4 strains of HIV-1.

Materials and methods

Engineering CD4+ T-cell lines stably expressing Tip, StpC,

or both Tip and StpC

MOLT4 cells were obtained from American Type Cul-

ture Collection and transduced to express Tip or StpC

using the murine stem cell virus (MSCV)-based gene

transfer system (Merlo et al., 1998). Briefly, MOLT4 cells

transduced with MSCV-based retroviral vectors driving the

expression of Tip or StpC and the corresponding control

vectors were cloned by antibiotic selection and subsequent

limiting dilution. Single-transductant clones were selected

for pac or StpC/pac expression with puromyocin (1 Ag/ml).

Clones expressing neo or Tip/neo were selected using

G418 (200 Ag/ml). ‘‘Double-transductants’’ were produced

by consecutively transfecting cells with two retroviral

vectors carrying different antibiotic selection markers.

Three cell types (neo/pac, neo/StpC-pac, and neo/Tip-

pac) were represented in this study by a single clone.

Tip+StpC+ cells (Tip-neo/StpC-pac) were represented by

two individual clones.

Generation of Tip/neo or StpC/pac pseudo-virus

Pseudo-viruses carrying the Tip/neo or StpC/pac genes

were produced as previously described (Hasham and

Tsygankov, 2004) and either used immediately or stored

at –80 jC.

Transduction of PBMCs and HUCLs with lentiviral vectors

driving Tip or StpC expression

PBMCs were prepared from normal blood donors using

Ficoll-hypaque (Pharmacia Biotech, Piscataway, NJ). Isolat-

ed PBMCs were cultured at densities between 1 � 106 and

2� 106 cells per milliliter in RPMI-1640 supplemented with

10% heat-inactivated fetal bovine serum and antibiotics

(complete medium). Human umbilical cord leukocytes were

isolated from umbilical cord blood using Ficoll-hypaque,

diluted in complete medium, and seeded in 24-well tissue

culture plates at a density of 1� 106 cells per milliliter. Plated

cells were transduced with 100 Al of lentiviral vector super-natants prepared as described in (Hasham and Tsygankov,

2004) to express either pac only, neo only, StpC and pac, or

Tip and neo. Following a 48-h period, the viral vectors were

removed, and cells were placed on antibiotic selection. Cells

transduced with neo-or tip/neo-vectors were grown in media

containing G418 (200 Ag/ml). Pac-or stpC/pac-transduced

cells were grown in media containing 1 Ag/ml of puromycin

and were added to the growth media. HUCL transductants

were simultaneously enriched for T-lymphocyte and mono-

cyte populations by adding cytokines IL-2 (100 ng/ml) and

M-CSF (100 ng/ml) to the HUCL culture media, respectively.

Transductants were placed on selection/enrichment for 7 days

before infection with either R5 or X4 strains of HIV-1.

Source and propagation of X4 and R5 strains of HIV-1

All handling of vector transductions were carried out in the

Biological Safety Level 2 facility at Temple University

School of Medicine using standard safety precautions. T-

tropic (X4) strains (IIIB and RF) and theM-tropic (R5) strains

(Ada-M, Ba-L, JRFL, and SF162) were obtained from the

AIDS Research and Reference Reagent Program (Division of

AIDS, NIAID, Rockville, MD). X4 strains were propagated

in MOLT4 4 cells, whereas primary cultures of normal

PBMCs were used to propagate R5 strains. To eliminate

soluble components, viruses were purified from culture

supernatants by ultracentrifugation at 33000 rpm for 1 h at

4jC in a Ti60 rotor. Pellets were collected, gently washed in

PBS, and resuspended in 1 ml complete medium per 50 ml of

supernatant. Concentrated virus was filtered through a 0.8-

Am filter and then stored at�70 jC. X4 HIV-1 was titered onSupT1 cells by syncytia formation. Stocks ranged from 106 to

107 syncytia-forming units per 1 ml. R5 strains were titered

using limiting dilutions followed by p24 analysis to deter-

mine the highest dilution giving rise to p24 values equal to 10

infectious units per milliliter.

Quantification of HIV-1 replication in Tip- or

StpC-transduced cell lines and primary lymphocytes

Syncytia formation

The infected-centers assay was used to measure syncytia

formation. Briefly, transduced cells were incubated with the

gy 324 (2004) 60–66

A.D. Raymond et al. / Virology 324 (2004) 60–66 65

virus at a multiplicity of infection of 1.0 at 37 jC for 2 h.

Infected cells were washed, serially diluted, and mixed with

2 � 105 SupT1 indicator cells. Syncytia were scored in

triplicate using an inverted scope 96 h post mixing with the

indicator cells.

Measuring p24 core antigen

Aliquots of infected cell culture supernatants were col-

lected at 4, 7, 14, and 21 days post infection and stored at

�80 jC. Viral concentrations were determined by a p24-

specific capture enzyme-linked immunosorbent assay

(ELISA) kit (SAIC-Frederick, Frederick, MD).

Immunoprecipitation and Western blotting

Immunoprecipitation and Western blotting to detect Tip

and StpC expression were performed as previously de-

scribed (Merlo et al., 1998). Briefly, StpC-transductants

were lysed in Tris/NaCl/EDTA (TNE) buffer containing

1% NP40 and 10 AM of aprotonin and leupeptin protease

inhibitors. StpC was detected by immmunoblotting of whole

cell lysates (100 Ag of total protein per lane). Lysates were

separated using SDS-PAGE and transferred to a nitrocellu-

lose membrane that was probed with anti-StpC sera. StpC

was then visualized using chemiluminescence visualized on

X-ray.

Tip was detected using the in vitro kinase assay. Cell

lysates containing 0.5–1 mg of total protein was subjected

to immunoprecipitation using anti-Tip sera and PANSOR-

BIN. Resulting precipitates were extensively washed and

then incubated in kinase buffer containing [g-32P] ATP

(NEN, Boston, MA) for 20 min. Reactions were stopped

by adding SDS-PAGE sample buffer. Phosphorylated pro-

teins were separated using SDS-PAGE and visualized by

autoradiography.

Statistical analysis

Statistical significance (P < 0.05) was determined using

Student’s t test comparisons between cells transduced with

either empty or Tip/StpC vectors.

Acknowledgments

This work was supported by grants RPG-00-105-01-

MBC and DHH5 5-R01-DA12113-03 from the American

Cancer Society and National Institute of Health, respective-

ly. We thank the NIH AIDS Research and Reference

Program for supplying reagents used in this study.

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