Herpes simplex virus type 2 infection increases human immunodeficiency virus type 1 entry into human primary macrophages
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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.
KeywordsU937 Cell Genital Ulcer Disease CCR5 Expression Level
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, 2, 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, 6, 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, 2, 3, 4, 5, 6, 7, 8, 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, 15, 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.
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