Herpes simplex virus type 2 infection increases human immunodeficiency virus type 1 entry into human primary macrophages
- Elena Sartori†1,
- Arianna Calistri†1,
- Cristiano Salata1,
- Claudia Del Vecchio1,
- Giorgio Palù1 and
- Cristina Parolin2Email author
© Sartori et al; licensee BioMed Central Ltd. 2011
Received: 18 November 2010
Accepted: 12 April 2011
Published: 12 April 2011
Epidemiological and clinical data indicate that genital ulcer disease (GUD) pathogens are associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and/or transmission. Among them, genital herpes simplex virus type 2 (HSV-2) seems to play a relevant role. Indeed, the ability of HSV-2 to induce massive infiltration at the genital level of cells which are potential targets for HIV-1 infection may represent one of the mechanisms involved in this process. Here we show that infection of human primary macrophages (MDMs) by HSV-2 results in an increase of CCR5 expression levels on cell surface and allows higher efficiency of MDMs to support entry of R5 HIV-1 strains. This finding could strengthen, at the molecular level, the evidence linking HSV-2 infection to an increased susceptibility to HIV-1 acquisition.
Herpes simplex virus (HSV), and especially HSV type 2, represents one of the most widely spread pathogen causing genital ulcer disease (GUD). Different studies have associated GUD aetiological agents in general, and HSV-2 in particular, with a higher risk to acquire and/or transmit HIV-1 infection [1–3]. A number of biological and molecular factors may explain this evidence. Both the physical disruption of the epithelial/mucosal barrier and the cellular inflammatory response characterizing GUD could facilitate HIV-1 acquisition, by providing the virus with access to a large number of CD4-positive cells. Moreover, several in vitro studies have underlined molecular mechanisms by which HSV can directly influence the HIV life cycle in HSV-HIV coinfected cells [2, 4]. Finally, randomised controlled trials have been conducted in coinfected individuals to evaluate the effect of HSV-2 suppressive therapy on HIV-1 genital shedding and plasma HIV-1 RNA, showing, in most cases, a negative impact on HIV-1 replication [5–7]. A recent study conducted by Zhu and co-workers  showed a persistence of HIV receptor-positive cells in genital skin after HSV reactivation. In the genital tract, macrophages represent one of the main target of HIV-1, especially during primary infection. In this study we wanted to analyze the ability of HSV-2 to infect human macrophages and to influence HIV-1 super-infection.
Thus, in order to analyze whether the low susceptibility to HSV-2 infection displayed by U937 cells could be related to their differentiation level, the cells were induced to differentiate by treatment with TPA (50 ng/ml). After twelve hours of incubation, U937 cells were washed twice with PBS and cultured for additional twenty-four hours in TPA-free medium, in order to avoid possible effects of residual TPA. The percentage of cells positive for CD14 surface expression, a marker of macrophage differentiation , was then determined by Fluorescence-Activated Cell Sorting (FACS). Briefly, 1 × 106 cells were harvested and directly incubated for one hour in cold PBS containing 1:100 (v/v) of an anti-human CD14 primary antibody (Li StarFISH). A fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz) was employed as secondary antibody and the fluorescence was evaluated by FACS analysis (FACScalibur, Beckton Dickinson). As reported in Figure 1B, after TPA treatment the percentage of CD14-positive U937 cells is significantly increased. Differentiated U937 cells were infected with HSV-2 (MOI of 1 PFU/cell). Infectious virus yields, which peaked approximately three days post-infection, appear to be significantly higher than those obtained from undifferentiated U937 cells (Figure 1C). Thus, our data suggest that HSV-2 replication efficiency is dependent on the differentiated phenotype of U937 cells along the monocytic pathway. Interestingly, while untreated U937 cells did not display a significant HSV-2 induced cytopathic effect (CPE), TPA-differentiated U937 cells were fully susceptible to viral CPE (data not shown).
In order to analyze whether the HSV-2 effect on CCR5 expression may have an impact on HIV-1 ability to enter macrophages, we employed a modified version of the previously described env-complementation assay, in which the HIV-1 envelope glycoprotein, expressed in trans, complements a single round of replication of an env-deleted provirus expressing the chloramphenicol acetyltransferase (CAT) gene [17, 18]. Since the defective HIV is capable of only one cycle of replication, this complementation assay allows us to quantitatively measure the abilities of the cells to support the entry of HIV-1 variants containing different envelope glycoproteins, by evaluating the level of CAT expression in the target cells. This assay represents an invaluable tool to dissect the contribution of viral/cellular determinants involved in HIV-1 entry [17, 18]. Recombinant HIV-1 viruses were produced by cotransfection of human embryonic kidney cells (293T, ATCC® Number: CRL-11268TM) with two plasmids, pSVCvpr+vpu+nef+Δenv-CAT and pSVIIIenv. The pSVCvpr+vpu+nef+Δenv-CAT is a derivative of the pSVC21, containing the HIV-1 HXBc2 molecular clone , where the vpu, vpr and nef sequences were substituted with those derived from the pNL4-3 (vpu/vpr)  and pLAI (nef)  molecular clones, in order to introduce functional vpu, vpr and nef genes. Starting from the pSVCvpr+vpu+nef+, we introduced by molecular biology techniques a 580 bp deletion (nucleotides 7041-8621) in the env gene and cloned the chloramphenicol acetyltransferase (CAT), obtained from the v653 RtatC vector , at the Bam HI site (nucleotide 8053) . The CAT gene is under the transcriptional control of the HIV-1 LTR and is expressed from a subgenomic mRNA generated by the same splicing events used for the natural HIV-1 nef message. Different pSVIIIenv plasmids encoding the HIV-1 Rev protein along with the envelope glycoproteins derived from laboratory-adapted T-cell-tropic (HXBc2), macrophage-tropic (JRF-L and ADA) and primary dualtropic (89.6) HIV-1 isolates, which can use CXCR-4 , CCR5  or either one  respectively, as a coreceptor, were adopted. Since the viral proteins are expressed in a context similar to that occurring in the authentic provirus, the levels of gene expression achieved are expected to resemble those in HIV-1-infected cells. Briefly, 293T cells were cotransfected by the calcium phosphate method with 20 μg of the pSVCvpr+vpu+nef+Δenv-CAT plasmid and 5 μg of pSVIIIenv plasmids expressing the HIV-1 HXBc2, ADA, JRF-L, or 89.6 envelope glycoproteins to produce recombinant virions. Control viruses lacking envelope glycoproteins were produced by transfecting 293T cells with the pSVCvpr+vpu+nef+Δenv-CAT plasmid alone.
Twelve hours post-transfection, 293T cells were washed and cultured in RPMI supplemented with 10% FBS. Conditioned medium containing recombinant viruses was harvested and filtered (0.45-μm-pore-size filter) twenty-four hours later. Recombinant viral particles were quantified by reverse transcription (RT) assay. Briefly, virions were precipitated from 1 ml of the filtered supernatants by centrifugation at 13,000 rpm for sixty minutes at 4°C. The precipitate was resuspended in 10 μl of a buffer containing 50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol (DTT), 20% glycerol, 250 mM KCl and 0.25% (v/v) Triton X-100, transferred in dry ice and lysed through three cycles of freezing and thawing. The sample was added to a reaction mixture containing 50 mM Tris-HCl pH 7.5, 7.5 mM MgCl2, 0.05% (v/v) Triton X-100, 5 mM DTT, 100 μg/ml polyA, 10 μg/ml oligo-dT and 2 μCi of 3H-dTTP (43 Ci/mmole) in a final volume of 50 μl. The reaction was incubated for one hour at 37°C and then transferred on Whatman filters. Filters were immediately washed three times in SSC 2× (0.3 M NaCl, 0.03 M sodium citrate pH 7.2) for 10 minutes each, twice in absolute ethanol for ten seconds each and then dried. The radioactivity was measured by using a scintillator (Rackbeta 1214 Wallac) and expressed in counts per million (cpm). In parallel, 1.5 × 106 of purified MDMs were cultured in complete RPMI containing GM-CSF in six-well plates for one week, before being infected with HSV-2 at the MOI of 10 PFU/cell, as previously described. Seventy-two hours later, the cells were transduced with 100,000 H3 cpm RT units of the different HIV-1 recombinant particles previously generated and expressing the CAT reporter gene. Seventy-two hours post-transduction, the MDMs were harvested, lysed in 150 μl of 250 mM Tris-HCl pH 7.5 and then assayed for CAT activity, as previously described . The different forms of acetylated chloramphenicol were separated by thin layer chromatography (TLC) and visualized with an autoradiografic exposure of twelve hours (Kodak Biomax films). The quantitative evaluation was obtained by cutting the TLC paper at the level of the corresponding spots, and by performing a quantification of the spots at the scintillator. The percentage of conversion in the acetylated forms was calculated as follows:
As mentioned above, interaction between HIV-1 and other sexually transmitted disease pathogens has been a subject of extensive investigation [1–9]. In particular, HSV-2 by affecting genital mucosa integrity and the function of cells physiologically forming a barrier against HIV-1 infection, such as Langherans cells , alters the cellular environment at the portal entry and facilitates HIV-1 acquisition/transmission . Significantly, in this study we demonstrate that HSV-2 infected macrophages, which represent one of the main target for HIV-1 infection at the genital mucosal site, display an enhanced expression of HIV-1 CCR5 coreceptor. This feature renders the cells more susceptible to infection especially by R5-tropic HIV-1 strains, which play a significant role in primary infection [14–16]. Human macrophages constitutively express HSV receptor HVEM (herpesvirus entry mediator)  and can be infected by HSV-2 [28, this report]. Taking into account this evidence and in vivo data demonstrating the enrichment of HIV receptor-positive inflammatory cells in the HSV-2 positive patient genitalia , our results describe one of the possible molecular mechanisms by which genital herpes may facilitate HIV-1 acquisition in HSV-2/HIV-1 coinfected patients.
We acknowledge Dr. Joseph Sodroski from Dana-Farber Cancer Institute, Harvard Medical School, for kindly providing the pSVIIIenv plasmids expressing the HIV-1 envelope glycoproteins derived from the ADA, 89.6, HXBc2 and JRF-L strains and the plasmid containing the HIV-1 HXBc2 provirus. We thank Paola Sette for technical help.
This work was supported by the MIUR-PRIN-2007 (#20072J9RWM_004 and 2007M52HTT_004) to CP and GP respectively, grants from the University of Padova (Ex-60%) to AC, GP and CP and from Istituto Superiore di Sanità (Rome-AIDS Project n. 30G.24, 30G.55 and 40G.44) to CP and GP.
- Ward H, Rönn M: Contribution of sexually transmitted infections to the sexual transmission of HIV. Curr Opin HIV AIDS 2010, 5: 305-310. 10.1097/COH.0b013e32833a8844PubMed CentralView ArticlePubMedGoogle Scholar
- Van de Perre P, Segondy M, Foulongne V, Ouedraogo A, Konate I, Huraux JM, Mayaud P, Nagot N: Herpes simplex virus and HIV-1: deciphering viral synergy. Lancet Infect Dis 2008, 8: 490-497. 10.1016/S1473-3099(08)70181-6View ArticlePubMedGoogle Scholar
- Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ: Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 2006, 20: 73-83. 10.1097/01.aids.0000198081.09337.a7View ArticlePubMedGoogle Scholar
- Palù G, Benetti L, Calistri A: Molecular basis of the interactions between herpes simplex viruses and HIV-1. Herpes 2001, 8: 50-55.PubMedGoogle Scholar
- Ouedraogo A, Nagot N, Vergne L, Konate I, Weiss HA, Defer MC, Foulongne V, Sanon A, Andonaba JB, Segondy M, Mayaud P, Van de Perre P: Impact of suppressive herpes therapy on genital HIV-1 RNA among women taking antiretroviral therapy: a randomized controlled trial. AIDS 2006, 20: 2305-2313. 10.1097/QAD.0b013e328010238dView ArticlePubMedGoogle Scholar
- Nagot N, Ouédraogo A, Foulongne V, Konaté I, Weiss HA, Vergne L, Defer MC, Djagbaré D, Sanon A, Andonaba JB, Becquart P, Segondy M, Vallo R, Sawadogo A, Van de Perre P, Mayaud P, ANRS 1285 Study Group: Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N Engl J Med 2007, 356: 790-799. 10.1056/NEJMoa062607View ArticlePubMedGoogle Scholar
- Zuckerman RA, Lucchetti A, Whittington WL, Sanchez J, Coombs RW, Zuñiga R, Magaret AS, Wald A, Corey L, Celum C: HSV suppression with valaciclovir reduces rectal and blood plasma HIV-1 levels in HIV-1, HSV-2 seropositive men: a randomized, double-blind, placebo-controlled crossover trial. J Infect Dis 2007, 196: 1500-1508. 10.1086/522523View ArticlePubMedGoogle Scholar
- Zhu J, Hladik F, Woodward A, Klock A, Peng T, Johnston C, Remington M, Magaret A, Koelle DM, Wald A, Corey L: Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med 2009, 15: 886-892. 10.1038/nm.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Calistri A, Parolin C, Pizzato M, Calvi P, Giaretta I, Palù G: Herpes simplex virus chronically infected human T lymphocytes are susceptible to HIV-1 superinfection and support HIV-1 pseudotyping. J Acquir Immune Defic Syndr 1999, 21: 90-98.PubMedGoogle Scholar
- Braun RW, Teute HK, Kirchner H, Munk K: Replication of herpes simplex virus in human T lymphocytes: characterization of the viral target cell. J Immunol 1984, 132: 914-919.PubMedGoogle Scholar
- Bruun T, Kristoffersen AK, Rollag H, Degré M: Interaction of herpes simplex virus with mononuclear phagocytes is dependent on the differentiation stage of the cells. APMIS 1998, 106: 305-314. 10.1111/j.1699-0463.1998.tb01351.xView ArticlePubMedGoogle Scholar
- Olsson IL, Breitman TR: Induction of differentiation of the human histiocytic lymphoma cell line U-937 by retinoic acid and cyclic adenosine 3':5'-monophosphate-inducing agents. Cancer Res 1982, 42: 3924-3927.PubMedGoogle Scholar
- Salata C, Curtarello M, Calistri A, Sartori E, Sette P, de Bernard M, Parolin C, Palù G: vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity. J Cell Physiol 2009, 219: 698-706. 10.1002/jcp.21722View ArticlePubMedGoogle Scholar
- Toma J, Whitcomb JM, Petropoulos CJ, Huang W: Dual-tropic HIV type 1 isolates vary dramatically in their utilization of CCR5 and CXCR4 coreceptors. AIDS 2010, 14: 2181-2186. 10.1097/QAD.0b013e32833c543fView ArticleGoogle Scholar
- Schuitemaker H, Koot M, Kootstra NA, Dercksen MW, De Goede REY, Van Steenwijk RP, Lange JMA, Eeftink Schattenkerk JKM, Miedema F, Tersmette M: Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus populations. J Virol 1992, 66: 1354-1360.PubMed CentralPubMedGoogle Scholar
- Stalmeijer EH, Van Rij RP, Boeser-Nunnink B, Visser JA, Naarding MA, Schols D, Schuitemaker H: In vivo evolution of X4 human immunodeficiency virus type 1 variants in the natural course of infection coincides with decreasing sensitivity to CXCR4 antagonists. J Virol 2004, 78: 2722-2728. 10.1128/JVI.78.6.2722-2728.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Helseth E, Kowalski M, Gabuzda D, Olshevsky U, Haseltine W, Sodroski J: Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants. J Virol 1990, 64: 2416-2420.PubMed CentralPubMedGoogle Scholar
- Parolin C, Gatto B, Del Vecchio C, Pecere T, Tramontano E, Cecchetti V, Fravolini A, Masiero S, Palumbo M, Palù G: New anti-human immunodeficiency virus type 1 6-aminoquinolones: mechanism of action. Antimicrob Agents Chemother 2003, 47: 889-896. 10.1128/AAC.47.3.889-896.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Ratner L, Haseltine W, Patarca R, Livac KJ, Starcich B, Josephs SJ, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, Ivanoff L, Petteway RR Jr, Pearson ML, Lauteenberger JA, Papas TS, Ghrayeb J, Chang NT, Gallo RC, Wong-Staal F: Complete nucleotide sequence of the AIDS virus, HTLV III. Nature 1985, 313: 227-283.Google Scholar
- Adachi A, Gendelman HE, Koenig S, Folks T, Wiley R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and non-human cells transfected with an infectious molecular clone. J Virol 1986, 59: 284-291.PubMed CentralPubMedGoogle Scholar
- Peden K, Emerman M, Montagnier L: Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology 1991, 185: 661-672. 10.1016/0042-6822(91)90537-LView ArticlePubMedGoogle Scholar
- Parolin C, Taddeo B, Palù G, Sodroski J: Use of cis - and trans -acting viral regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes. Virology 1996, 222: 415-422. 10.1006/viro.1996.0438View ArticlePubMedGoogle Scholar
- Feng Y, Broder CC, Kennedy PE, Berger EA: HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996, 272: 809-810. 10.1126/science.272.5263.872View ArticleGoogle Scholar
- Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J: The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996, 85: 1135-1148. 10.1016/S0092-8674(00)81313-6View ArticlePubMedGoogle Scholar
- Collman R, Balliet JW, Gregory SA, Friedman H, Kolson DL, Nathanson N, Srinivasan A: An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol 1992, 66: 7517-7521.PubMed CentralPubMedGoogle Scholar
- de Jong MA, de Witte L, Taylor ME, Geijtenbeek TB: Herpes simplex virus type 2 enhances HIV-1 susceptibility by affecting Langerhans cell function. J Immunol 2010, 185: 1633-1641. 10.4049/jimmunol.0904137View ArticlePubMedGoogle Scholar
- Corey L, Wald A, Celum CL, Quinn TC: The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004, 35: 435-445. 10.1097/00126334-200404150-00001View ArticlePubMedGoogle Scholar
- Taylor JM, Lin E, Susmarski N, Yoon M, Zago A, Ware CF, Pfeffer K, Miyoshi J, Takai Y, Spear PG: Alternative entry receptors for herpes simplex virus and their roles in disease. Cell Host Microbe 2007, 2: 19-28. 10.1016/j.chom.2007.06.005PubMed CentralView ArticlePubMedGoogle Scholar
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