Summary:

Peripheral blood lymphocytes (PBL) and CEM CD4+ T-cell line can be infected by herpes simplex virus-1 (HSV-1). CEM cells were characterized as a cellular model to study interactions occurring between HSV-1 and HIV-1. Virtually all cells were persistently infected by HSV-1 (CEM

HSV) and expressed the latency

associated transcripts, whereas only a fraction tested positive for HSV-antigens. CD4 and CXCR-4 expression and function were not affected in CEMHSV cells and no

significant increase of deoxyribonucleotide pools was noticed. Superinfection of CEMHSV cells with HIV-1 led to a cell line chronically infected by both viruses

(CEMHIV/HSV). Evidence was also obtained that this cell line can produce HIV-1 pseudotyped by HSV-1 envelope. These results may have important implications for a

better under-standing of AIDS pathogenesis.

Individuals infected by HIV-1 often harbor infection by a number of other viruses, such as HTLV I/II, papovaviruses, hepatitis B and C viruses (HBV, HCV), human

cytomegalovirus (HCMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus types 1 and 2 (HSV-1/2), human herpesviruses 6, 7, and 8 (HHV-

6/7/8). Opportunistic viral agents, among them herpesviruses, have proved to influence AIDS progression and patient survival (1). HSV can cause severe clinical

manifestations in HIV-1-infected individuals, particularly during late stages of disease. At variance with immunocompetent individuals, mucocutaneous

manifestations in AIDS patients are represented by herpetic vesicles that progress to extensive ulcerations, involving larger areas of skin and deep tissues.

Occasionally, HSV may also spread from the initial lesion to other organs by systemic dissemination causing life-threatening diseases. In addition to their behavior as

opportunistic pathogens, HSV were suggested to interact with HIV-1 at molecular and cellular levels, without causing any clinical disease but promoting the

pathogenesis of AIDS (2,3). In this regard, HSV-induced transactivation of HIV-1 long terminal repeat (LTR) has been shown to stimulate expression of HIV-1 genes in

cells chronically infected by HIV-1 (4-8). The proposed mechanism involves both HSV-induced activation of cellular factors, like NF-[kappa]B, and a direct HIV-1 LTR

transactivation mediated by the virus-encoded immediate-early protein ICP0 (7,8). Considering the in vivo occurrence of HSV/HIV interactions, several clinical

studies have also addressed the role of antiherpetic therapy in delaying AIDS progression (9,10) and evidence was provided that HSV-2-seropositive individuals and

those with genital herpes may have a higher risk of transmitting and acquiring HIV-1 infection (11,12). HSV may also extend the range of cells susceptible to HIV-1,

as indicated by an electron microscopy observation showing that CD4- epidermal keratinocytes, obtained from HIV/HSV-seropositive patients, contained high

numbers of HSV-1 and HIV-1 particles (13). This finding supports the hypothesis that HIV-1 can be pseudotyped by HSV glycoproteins, thus infecting also CD4- cells.

Such a phenomenon has previously been shown to occur in hamster cells (CHO), which enabled the growth of a virus representing a phenotypic mixture between

wild-type HIV-1 and a ts mutant of HSV-1 strain (14).

For all these considerations, it would be critical to ascertain whether an interaction between HSV-1 and HIV-1 takes place in CD4+ lymphocytes, because these

cells are the natural target of HIV-1 infection in vivo and may also represent a reservoir for HSV-1 replication and spreading in humans (15-21). In this report, we

explored the ability of HSV-1 to infect human peripheral blood lymphocytes (PBL) and CD4+ human lymphoblastoid cells. In particular, we propose the use of CEM

cells as an in vitro model to study molecular interactions occurring between HIV-1 and HSV-1, among which pseudotyping that may be relevant to the pathogenesis

of AIDS.

METHODS

Cells and Viruses CCRF-CEM (CEM) cells, CEM-SS cells (CEM-derived cells adapted to support HIV-1 growth and syncytia formation), CEMHSV cells (discussed later), H9 cells

chronically infected by HIV-1 IIIB, and uninfected H9 cells were grown in Roswell Park Memorial Institute medium (RPMI-1640 culture medium) with addition of 10%

inactivated fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml) and glutamine (2 mM). PBL from healthy donors were isolated from heparinized

blood by Ficoll-Hypaque (Histopaque 1077, Sigma, St. Louis, MO, U.S.A.) gradient centrifugation. Mononuclear cells at the interface were collected and washed

three times with RPMI-1640 and resuspended in RPMI-1640 supplemented with 10% FCS. They were then cultured at 37°C in 5% CO2 with or without interleukin-2 (IL-

2, 10 U/ml) and phytohemagglutinin (PHA, 1% v/v). African green monkey kidney cells (Vero) and COS-1 cells were grown in Dulbecco's modified Eagle's medium

(DMEM) added as described above. CEM, CEMHSV, and CEM-SS cells were negative for HHV-6, HHV-7, HHV-8, and Mycoplasma infection. HSV-1 strain 17 was kindly

provided by the MRC Virology Unit, Institute of Virology, Glasgow, U.K. Virus was grown and titered by plaque reduction on Vero cells monolayers as previously

described (22). PBL were infected with HSV-1 stock at a multiplicity of infection (MOI) of 1 plaque forming unit (PFU)/cell for 90 minutes at room temperature. Cells

were washed three times with RPMI-1640, resuspended in medium containing 10% FCS, and cultured at 37°C with or without PHA (1% v/v) and IL-2 (10 U/ml). CEM

cells were initially infected with HSV-1 at a MOI of 1 PFU/ml for 90 minutes at room temperature, washed, and subsequently incubated at 37°C. These cells were

maintained indefinitely in culture and characterized as CEMHSV

cells (see Results). Infectious virus was obtained both from culture supernatant and from cell lysates

after three cycles of freeze-thawing and sonication in ice. To inhibit HSV-1 infection, human immunoglobulin G (IgG) with a HSV-1 microneutralization titer of 1:50

v/v was added to a final concentration of 1:10 v/v. HIV-1 IIIB was obtained from chronically infected H9 cells after coculture with freshly grown uninfected H9 cells

in a 1:4 ratio. Virus was titered on CEM-SS cells, and reverse-transcriptase (RT) activity was measured as previously described (23). Levels of HIV-1 p24 antigen were

determined by HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) kit (NEN Life Science Products, Boston, MA, U.S.A.), according to the manufacturer's

instructions. HIV-1 infection of CEMHSV cells was conducted for 2 hours at 37°C. Cells were then washed and further incubated in complete medium.

Herpes Simplex Virus Chronically Infected Human T Lymphocytes Are Susceptible to HIV-1 Superinfection and Support HIV-1 Pseudotyping

Keywords: HSV-1, HIV-1, Superinfection, CD4, CXCR4, Deoxyribonucleotide pools, Pseudotyping

ISSN: 1525-4135

Accession: 00126334-199906010-00003

Author(s): Calistri, Arianna; Parolin, Cristina; Pizzato, Massimo; Calvi, Paola; Giaretta, Ilaria; Palù, Giorgio

Issue: Volume 21(2),�1 June 1999,�pp 90-98

Publication Type: [Basic Science]

Publisher: © 1999 Lippincott Williams & Wilkins, Inc.

Institution(s):

Institute of Microbiology of the University of Padova, Padova, Italy

Address correspondence and reprint requests to Giorgio Palù, Institute of Microbiology of the

University of Padova, via A. Gabelli 63, 35121 Italy; email: gpalu@uxl.unipd.it.

Manuscript received December 28, 1998; accepted February 18, 1999.

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Syncytia Formation Assay

COS-1 cells were transfected by the diethylaminoethyl (DEAE)-dextran method (23) with 10 µg of the pCMVenv[DELTA]Xho plasmid, containing env and rev genes

of HIV-1 HXBc2 under control of the constitutive IE promoter of CMV (23). Medium was changed 48 hours after transfection. The following day, cells were detached

from the plate with 50 mM ethylenediaminetetraacetic acid (EDTA) pH 7.5 and mixed with CEM cells or CEMHSV cells in a 1:10 ratio. The mixed culture was

incubated at 37°C for 8 to 12 hours in RPMI-1640 containing 10% FCS and 1 µM ganciclovir (GCV, Glaxo-Wellcome, Beckenham, U.K.) to prevent infection of COS-1

cells by HSV-1. Syncytia formation was observed and scored by light microscopy.

Polymerase Chain Reaction and Quantitative Analysis of Deoxyribonucleotide Pool

Detection of HSV-1 and HIV-1 DNA was performed by polymerase chain reaction (PCR) on total cellular DNA extracted from 106 cells. Amplification was carried

out as follows: primers specific for the viral tk gene (HSV-A1 [47635-47656] and HSV-A2 [47419-47436]) were used in a 35-cycle reaction to detect HSV-1 DNA

(denaturation step: 1 minute at 94°C; annealing step: 1 minute at 53°C; elongation step: 1 minute at 72°C); LR33 (6972-6993) and LR34 (7367-7387) primers,

mapping in the viral env gene, were used to detect HIV-1 proviral DNA in a 40-cycle reaction (denaturation step: 1 minute at 94°C; annealing step: 1 minute at 56°

C; elongation step: 1 minute at 72°C). In both cases, a final extension step of 10 minutes at 72°C was carried out, and the amplified DNA products were subjected

to electrophoretic analysis in a 2% w/v agarose gel. Deoxyribonucleotide phosphates (dNTPs) were extracted from 107 HSV-1-infected and mock-infected cells (Vero,

CEM, and PBL) with 60% v/v ice-cold methanol for 30 minutes, as previously described (24). Quantitative analysis of dNTP pool was performed by measuring the

extent of [3H]-deoxyadenosine triphosphate (dATP) incorporation into synthetic oligonucleotides using Klenow fragment DNA polymerase, according to the method

proposed by Sherman and Fyfe (25).

Immunofluorescence and in Situ Hybridization To assess the number of virus-producing cells, PBL and CEMHSV cells were washed with phosphate buffered saline (PBS), cytospun by low-speed centrifugation on

glass slides, and fixed in cold acetone/methanol 1:1 v/v for 5 minutes. Fixed cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated polyclonal

anti-HSV-1 antiserum (DAKO, Glostrup, Denmark), which detects all major HSV-1 glycoproteins. The same analysis was performed with a series of antisera reacting

against the following HSV-1 proteins: ICP0 (kindly provided by H. Marsden, MRC Virology Unit, Glasgow, Scotland, U.K.), ICP6 (kindly provided by H. Marsden), ICP36

(kindly provided by E. Blair, Glaxo-Wellcome U.K.), glycoprotein D (mAbE-II-24G10 kindly provided by S. Welling-Wester, University of Groningen, Groninger, the

Netherlands) and US11 (kindly provided by B. Roizman, University of Chicago, IL, U.S.A.). Flow cytometric analysis of CEM and CEMHSV

cells was conducted with a

Bryte-HS flowcytometer using the Win-Bryte software (BioRad Microscience Ltd., Hemel Hempstead, U.K.). For this assay cells were washed with PBS and then fixed

with 75% ethanol at -20°C for 20 minutes. After three more washings with PBS and resuspension of cells in 100 µl PBS, 10 µl polyclonal anti-HSV-1 antiserum, 10 ml

Leu3A anti-CD4 monoclonal antibody (Becton-Dickinson, Meylan, France) and 10 µl 12G5 anti-CXCR-4 monoclonal antibody (Pharmingen, San Diego, CA, U.S.A.) were

added separately and cells were incubated for 30 minutes at room temperature. For in situ hybridization (ISH), CEM and CEMHSV cells were washed, deposited by low

speed centrifugation onto a glass slide, and fixed in 100% ethanol. The RNA probe used for this assay was obtained by T7 polymerase transcription of the HindIII-

linearized pS-LAT2 plasmid. The pSLAT2 plasmid contains the region spanning from nucleotide 118867 to nucleotide 120301 of the LAT sequence of HSV-1 strain 17,

cloned between T7 and T3 promoters (26). The digoxigenin (DIG) detection system was used for assessing the presence of a positive signal, as previously described

(26).

RESULTS

Peripheral Blood Lymphocytes Infection With HSV-1

For confirming the possibility that HSV-1 could infect human lymphocytes (15-17,19,20), PBL were isolated by healthy donors and infected with HSV-1 stock at a

MOI of 1 PFU/cell. Cells were then cultured with or without PHA (1% v/v) and IL-2 (10 U/ml) for a variable length of time. Infection was monitored by

immunofluorescence analysis, with antibodies directed against immediate early (ICP0), early (ICP6 and ICP36), and late (gD, US11, pool of glycoproteins) antigens of

HSV-1. Results (Fig. 1A; Table 1) proved that PBL are infectible by HSV, with ~20% of cells being positive for viral antigens. No discordance was determined in the

expression of the three antigen classes, indicating that IF+ cells were productively infected. However, infection was possible only when PBL were activated by

mitogen treatment with mitogens (Fig. 1A; Table 1), given that no viral antigen expression was detectable in nonstimulated cells (Fig. 1B; Table 1).

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FIG. 1. Immunofluorescence analysis of peripheral blood lymphocytes infected with HSV-1 (A) with or (B) without PHA (1% v/v) and IL-2 (10 U/ml) stimulation. For

this assay, a FITC-conjugated polyclonal antiserum that detects all major HSV-1 glycoproteins was used. Negative cells were stained with blue Evans dye (0.01% v/v).

TABLE 1. Expression of HSV-1 antigens in PBLs and in CEMHSV cells at different time points after viral infection (pi)

Biologic and Molecular Characterization of CEM Cells Infected With HSV-1 (CEMHSV

)

Unlike PBL, CEM cells can be infected with HSV-1 without mitogenic activation and can be maintained in vitro for a long time. When CEM cells were infected

with HSV-1 at a MOI of 1 PFU/ml, production of infectious virus resulted in a peak of 0.16 ± 0.08 PFU/cell at 48 hours postinfection (pi). Thereafter a plateau of

0.06±0.02 PFU/cell was reached, with 5% to 20% of cells being positive for viral immediate early (ICP0), early (ICP6 and ICP36), and late (gD, US11, pool of

glycoproteins) antigens according to the technique in use (immunofluorescence or flow cytometry; Table 1; Fig. 2A). PCR was always positive for HSV-1 DNA, even at

a single cell dilution (not shown). A significant increase in the production of infectious virus could be elicited by cells stimulated with 1% v/v PHA, accounting for an

infectious yield of 0.5±0.05 PFU/cells. In situ hybridization, performed on CEMHSV cells with a probe specific for the latency associated transcripts (LAT), allowed to

appreciate that almost 95% of CEMHSV

cells expressed LAT mRNAs at high levels in the cytoplasm (Fig. 2B). These results suggest that HSV-1 is present in most CEM

cells, probably in a classical latent state. Productive infection must be a constant breakthrough of infectious virus in a fraction of cells.

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FIG. 2. (A) Immunofluorescence (left) and flow cytometric analysis (right) of CEMHSV cells. For this analysis, a polyclonal anti-herpes simplex virus type 1 antiserum

was used. (B) In situ hybridization of CEM (left) and CEMHSV (right) cells with a RNA probe specific for the LAT transcripts.

HSV-1 Chronic Infection Does Not Affect CD4 and CXCR-4 Expression and Function in CEM Cells

HIV-1 infection of T cells is mediated by CD4, which is the major viral receptor. The presence of this molecule is essential but not sufficient for HIV-1 entry,

inasmuch as HIV-1 coreceptors are needed (27,28). Because some herpesviruses can modulate expression of HIV receptors and coreceptors (29-32), CEMHSV cells

were examined for CD4 and CXCR-4 expression, the latter being the HIV-1 coreceptor on the T-cells' surface. Data obtained by flow-cytometry analysis indicated

that these molecules are expressed at a comparable level in CEM and CEMHSV cells (Fig. 3A). CD4 and CXCR-4 functional activity was also comparable in both cell

populations, as shown by a syncytia formation assay (Fig. 3B) in coculture with cells expressing the HIV-1 envelope. These results ruled out the possibility that HSV-1

can alter CEM cells' susceptibility to HIV-1 superinfection at the level of the HIV-1 binding/fusion steps.

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FIG. 3. Chronic herpes simplex virus type 1 infection did not modify expression and function of CD4 and CXCR-4. (A) Flow cytometric analysis of CEM

HSV and CEM

cells for the expression of CXCR-4 (left) and CD4 (right) antigens. (B) Syncytia formation assay. CEMHSV

and CEM cells were cocultured with COS-1 cells transfected

with the pCMVenv[DELTA]Xho plasmid (+Env), or mock-transfected (-Env), in a 1:10 ratio. Syncytia were scored per microscopic field, considering only those cells

that were at least twice the size of the biggest cell in the control field ± standard error of the mean of 10 random fields.

Quantitative Analysis of Deoxyribonucleotide Pools in PBL and CEM Cells Infected With HSV-1

An important biologic parameter for the fate of HIV-1 infection in target cells is the presence of a high intracellular concentration of dNTPs (33-35). Therefore,

it was relevant to study the effect of HSV-1 infection on lymphocyte dNTP pools. At variance with Vero cells, which are fully permissive to HSV replication, the

deoxythymidine triphosphate (dTTP) pool size was only moderately increased in CEM cells following HSV-1 infection (1.1-fold compared with 20-fold). Similar results

were obtained in PHA- and IL-2-stimulated PBL infectable with HSV-1 (Table 2). Thus, the dNTP pool increase that was shown to occur in Vero cells infected with

HSV-1 was not observed in lymphocytes and in lymphoblastoid cells. This result indicates that the activity of viral enzymes involved in dNTP synthesis, such as

ribonucleotide reductase (ICP6) and thymidine kinase (ICP36), is not sufficient to modify the dNTP basal levels significantly (Table 2). This rules out a role for HSV in

modulating the dNTP pool in lymphocytes.

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TABLE 2. dNTP pools in cells infected with HSV-1 (MOI = 5 PFU/cell) at different time points postinfection (pi)

HIV-1 Superinfection of CEMHSV

and HIV-1 Pseudotyping by the HSV-1 Envelope

CEMHSV

cells might represent a useful model to evaluate potential HIV-HSV interactions occurring at the cellular level. CEMHSV

cells were superinfected by HIV-

1, leading to a cell line carrying a double chronic infection that was stable with time in culture (CEMHSV/HIV

). Maintenance of both viral genomes was confirmed by

PCR, 90 days after superinfection with HIV-1 (Fig. 4). Production of HIV-1 and HSV-1 infectious particles were observed in CEMHSV/HIV

cells. When CD4- Vero cells

were infected with the CEMHSV/HIV cell-free supernatant, production of p24 HIV antigen was also observed (~300 ng/ml). In addition, p24 was detectable during the

first 48 hours of infection, before a clear HSV-1-related cytopathic effect was evident. PCR analysis confirmed the presence of HIV-1 proviral DNA in these cells (Fig.

5). By contrast, no sign of HIV-1 infection was noticed when Vero cells were exposed to CEMHSV/HIV cell-free supernatant, in the presence of human HSV-1

neutralizing IgG or of 3'-azido-3'deoxythymidine (AZT; Fig. 5). These results clearly indicate that some of the infectious virions entering Vero cells contain an HIV-

genome, capable of expression. HIV-1 infection of these cells is possible, as shown by neutralization experiment, only if the HIV-1 core particles are pseudotyped by

an HSV envelope.

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FIG. 4. HIV-1 superinfection of CEMHSV cells led to a cell line chronically infected by both viruses (CEMHSV/HIV). Polymerase chain reaction was performed on total

DNA extracted from the indicated cells. DNA from doubly infected cells was extracted 90 days after infection of CEMHSV cells with HIV-1. Primers for HSV-1 mapped

within the tk gene, whereas primers for HIV-1 mapped within env gene (see Methods).

FIG. 5. HIV-1 derived from CEM

HSV/HIV cells is capable of infecting CD4- Vero cells, as assessed by the presence of proviral DNA. Lane 1: molecular weight marker.

Lane 2: polymerase chain reaction (PCR)-negative control. Lane 3: PCR positive control. Lane 4: Vero cells infected with supernatant of H9 cells chronically infected

by HIV-1. Lane 5: Vero cells infected with cell-free supernatant of CEMHSV/HIV cells. Lane 6: the same as in lane 5 with addition of 3.7-µM AZT. Lane 7: the same as

in lane 5 with addition of human herpes simplex virus type 1-neutralizing IgGs at the dilution of 1:10 v/v. For inoculation into Vero cells, phenotypic mixtures and

the HIV-1 controls were adjusted to equivalent amounts of input HIV-1 their content of p24 antigen.

DISCUSSION Besides causing severe opportunistic infections at late stages of AIDS, HSV have been implied as cofactors in AIDS pathogenesis, that is, as agents able to

interact with HIV-1 at cellular and/or molecular levels to promote the lentiviral infection (2,3). Complex biologic phenomena such as interactions between HSV-1

and HIV-1 need to be investigated in an appropriate in vitro system, mimicking the in vivo environment wherein the two viruses most likely interact. In this paper,

data are presented showing that the human lymphoblastoid CEM cell line can be persistently infected with HSV-1 (CEMHSV). CEMHSV cells, in turn, retain

susceptibility to HIV-1 superinfection and support HIV-1 pseudotyping by HSV-1 envelope. Carrying a dual chronic viral infection (CEMHSV/HIV

), these cells may

represent a useful model system to study HSV-HIV interactions at the molecular level. The CEM cell line was adopted after demonstration that HSV-1 could infect

primary human lymphocytes, although with a low efficiency, provided these cells are stimulated with PHA and IL-2. This finding, confirming almost obsolete reports

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in the literature (16,19), prompted us to search for a human lymphoblastoid T-cell line susceptible to HSV-1 infection, with the advantage, over primary

lymphocytes, of an indefinite in vitro life. One of the main observation of our investigation is that CEMHSV

cells have the property of harboring HSV-1 in a

semirepressed state, with only a fraction of cells (5%-20%) staining positive for viral late antigens at any given time point. This condition leads to production of low

titer infectious virus. PCR-limiting dilution experiments confirmed that every cell contained an HSV genome. As revealed by in situ hybridization, almost all CEMHSV

cells (>95%) were strongly positive for LAT transcripts, with an atypical intracytoplasmic localization. The latter observation confirms that at least one viral genome

per cell was present. Thus, productive infection would be the result of a steady state transition from viral latency to viral reactivation. This is the first description

of LAT in HSV-1-infected lymphocytes, although cytoplasmic localization was previously reported in infected neuroblastoma and in CV-1 cell lines (36,37). The likely

explanation for this atypical finding must be related to the replicative state of cells in culture, differing from that of postmitotic neuronal cells. Our demonstration

that CEMHSV

cells abundantly express LAT could be significant for timing of HSV-1 infection in lymphoblastoid cells. Moreover, this result supports previous evidence

that HSV-1 infects lymphocytes in vivo and strengthens the possibility that these cells may act as a site of latent infection and reactivation (38). Having elucidated

the relative competence of CEM cells for HSV replication, we examined potential mechanisms by which HSV-1 could affect CEM susceptibility to HIV-1 infection.

Expression of CD4 and CXCR-4 was not modified in CEM cells following chronic infection by HSV-1, at variance with the effects produced on lymphocytes by other

herpesviruses (29-32). This observation, revealed by cytofluorimetric analysis and confirmed by syncytiaformation assay, ruled out the possibility that HIV-1

superinfection of CEMHSV could be promoted or inhibited at the level of the lentivirus binding or internalization. Unlike other cellular systems (39), HSV-1 infection

failed to modify the pool of dNPTs both in PBL and in CEM cells, a reflection that HSV-1[beta] genes are only partially expressed in a minority of these cell

populations. Enhancement of dNTP pools in activated PBL has been shown to stimulate HIV-1 replication by increasing the processing of reverse transcriptase and

the consequent formation of the full-length proviral DNA (33-35). Consistent with this observation, HIV-1 replication was repressed in activated PBL and T-cell lines

by inhibition of cellular ribonucleotide reductase (39). In accord with these arguments, the presence of a modified dNTP pool in CEMHSV

excludes the occurrence of

a positive metabolic cooperation between HSV-1 and HIV-1, at least in this model system. A crucial implication for AIDS pathogenesis, emerging from our study, is

the possibility that CEM cells harbor a dual chronic infection by HSV-1 and HIV-1 (CEMHSV/HIV), a condition that may allow a phenotypic mixture of the two viruses.

The phenomenon by which a virus acquires the envelope of a different virus by infecting the same cell is known as pseudotyping. Although HIV-1 can be pseudotyped

by various other envelopes (40,41), evidence that this could occur through HSV-1 is rather indirect so far, being confined to an ultrastructural morphologic

examination (13) in keratinocytes and to the use of a genetically modified HSV-1 in hamster CHO cells (14). In this paper, we present the first direct evidence that

HIV-1 pseudotyping by an wild-type HSV-1 envelope can take place in lymphocytes. The model used (CEMHSV/HIV) has in vivo relevance because T lymphocytes are

the major target cells of HIV-1 as well as the largest reservoir of this virus. Hence, if an interaction between HIV-1 and HSV-1 occurs in vivo at the cellular level, it is

most likely to occur in lymphocytes, as would be in the case of genitomucosal herpetic lesions where high numbers of infiltrating, activated T lymphocytes are

always present. Activated T cells and their environment, besides providing substrate and conditions that favor a higher transmission and/or acquisition of HIV-1

infection (11,12), could serve to spread both HSV-1 and HIV-1 by chronic carrier state and by pseudotyping, as shown by our results. In particular, pseudotyping

could be extremely relevant to AIDS progression, in that a recombinant virion containing the HIV-1 core and an HSV-1 envelope could infect a large spectrum of CD4-

cells, spreading HIV-1 infection to different organs. Such a phenomenon could explain the presence of an elevated HIV-1 load in the absence of circulating CD4 cells

in terminal phases of AIDS. In conclusion, we present a cellular model that supports the possibility that HIV-1/HSV-1 can interact in CD4 T lymphocytes. Our model

mechanistically confirms previous data showing that HSV-1 stimulates HIV-1 replication and gene expression in cell lines chronically infected by HIV-1 (4-8). Both

these phenomena would occur by an HIV-LTR transactivation mechanism, mediated by the HSV-1 proteins ICP0 and ICP27 (7). Besides, HSV-1 infection was shown to

induce cellular factors, like NF-[kappa]B, that interact positively with the HIV-1 LTR (8). An additional positive interaction between HSV-1 and HIV-1 could be driven

by the HSV-1 Us11 late protein which, binding Rex- and Rev-responsive elements, is able to transactivate envelope retroviral glycoprotein expression (42). A chronic

carrier state of HSV-1 and HIV-1 in lymphocytes and HIV-1 pseudotyping are phenomena that may be relevant to AIDS pathogenesis. However, the question whether

the above interactions (as well as others) can occur in vivo remains unanswered. For this reason, studies should specifically address this point also in the context of

evaluating the efficacy of antiviral therapy.

Acknowledgments: This work was supported by the Istituto Superiore di Sanità of Italy, AIDS Project (n 40 A.0.76), MURST 40-60%, Biomed 2, Regione Veneto

and Fondazione Cassa di Risparmio di Padova e Rovigo. We thank Roberta Bonaguro and Teresa Pecere for their suggestions and critical revision of the manuscript.

Financial support to G. Palù was provided by Istituto Superiore di Sanità of Italy, AIDS Project (n 40 A.0.76), MURST 40-60%, Biomed 2, Regione Veneto and

Fondazione Cassa di Risparmio di Padova e Rovigo.

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Key Words: HSV-1; HIV-1; Superinfection; CD4; CXCR4; Deoxyribonucleotide pools; Pseudotyping

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