Herpes simplex virus type 2 infection. Part 3
Thus, R5-tropic strains appear to be the most relevant during HIV-1 acquisition and early phases of infection. Taking into account all these considerations, we focused our attention on HIV-1 CD4 receptor and on the CCR5 coreceptor. 2 × 106 purified MDMs were infected with HSV-2 at the MOI of 10 PFU/cells and, seventy-two hours later, the cells were harvested and analyzed by FACS for the level of CD4 and CCR5 surface expression. In particular, either a CD4 (1:200 v/v, BD Pharmingen) or a CCR5 (1:50 v/v, BD Pharmingen) specific primary antibody, along with a FITC-conjugated anti-rabbit immunoglobulin G secondary antibody (Santa Cruz), were employed on fresh MDMs. Our data show that the percentage of both CD4 and CCR5 positive cells increases after HSV-2 infection (Figure 2B) (p < 0.05). At the same time, also the expression levels of HIV-1 receptor and coreceptor on the cell surface is slightly enhanced, as indicated by the shift in the mean fluorescence intensity displayed by the FACS histograms.
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. 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. 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 [19], 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 BamHI 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.