Colocalization of HSV-2 gB and gD with gp160 in co-infected or transfected cells
Prior evaluating the potential formation of pseuotyped particles, we first analyzed at the cellular level the co-expression of gB and gp160, or gD and gp160, in HIV-1 infected P4P cells superinfected by HSV-2, and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD, by using confocal microscopy. Our observations confirm that P4P cells exposed to both HIV-1 and HSV-2 can be co-infected and coreplicate the two viruses, since we can detect in the same cell glycoproteins from both viruses. HIV-1 P4P cells transfected with plasmids coding for gB or gD also express both the HSV-2 glycoprotein and gp160 (Figure 1). In addition, as shown in Figure 1, the superposition of fluorescence signals indicates that gB and gD may co-localize with HIV-1 gp160 in both co-infected and transfected P4P cells. The pattern of staining in single infections with either HIV-1 or HSV-2 was the same as that observed in co-infected cells (not shown).
CEM cells are susceptible to HSV-2 and HIV-1 co-infection
Prior HSV-1 infection of CEM cells has been shown to result in a delay of HIV-1 production, due to cell growth inhibition and apoptosis, while HSV-1 superinfection of HIV-infected CEM cells promoted HIV expression . We have therefore focused our analysis on this latter condition.
CEM cells infected by HIV-1NDK strain were superinfected by HSV-1 or HSV-2 at a multiplicity of infection (MOI) of 0.01, 0.1 or 1 pfu/cell for 72 h. Every each 24 hours, culture supernatant was collected for viral quantifications by real time PCR, and after washing cells were reincubated with fresh medium. Our results show that HSV-1 and HSV-2 did not modify the production of HIV-1 RNA, and that HIV-1 did not influence either HSV-1 or HSV-2 replication in CEM cells (Figures 2 and 3). The cell free supernatants of dually infected CEM cells were then used for the infection of Vero cells. In the first 48 h, cytopathic effect related to HSV-2 was observed by optical microscopy (not shown). No HIV-1 p24 antigen was detected in the supernatant of Vero cells at 24 h or 48 h, and DNA of the Vero cell pellet contained no HIV-1 proviral DNA as detected by real time PCR (not shown). The HSV-1 and HIV-1- co-infected CEM cells and HSV-2- and HIV-1- co-infected CEM cells did not lead to the production of HIV-1 virions able to infect cells that are usually non-permissive.
HIV-1 particles produced from HIV-1 and HSV-2 co-infected epithelial cells do not infect Vero cells
Intestinal HT29 cells are epithelial cells susceptible to infection by both R5 and X4 HIV-1 strains . HT29 cells were infected with HIV-1NDK for 48 h before HSV-2 superinfection at MOIs of 0.01, 0.1 and 1. HIV-1 production was assessed at 24 and 48 h following the infection. At 24 h, the medium was removed for viral quantification and was replaced with fresh medium for the 48 hour time-point. The results depicted in Figure 4 show that HIV-1 production by HT29 cells decreased significantly following HSV-2 superinfection at a MOI of 1 pfu after 24 h (P = 0.04). The influence of HSV-2 at a MOI of 1 pfu on the decrease of HIV replication was more pronounced 24 hours later. In contrast, lower HSV-2 inoculum (0,1 and 0.01 pfu) did not seem to inhibit HIV-1 production in a 48 h delay. Supernatants of HIV-1 and HSV-2 dually infected HT29 cells collected at 24 and 48 h did not lead to HIV-1 infection of Vero cells as determined by detection of proviral DNA, while HSV-2 replication was confirmed by the detection of typical cytopathic effect (not shown).
P4P cells, derived from the endometrial epithelium, were also infected in the same conditions as those used for HT29 cells. The rate of HIV-1 infection of P4P cells was around 50%, as determined by the ratio of HIV-1 DNA and albumin levels, suggesting that any viral superinfection would probably produce dually infected cells. As shown in Figure 5, superinfection by HSV-2 did not significantly influence HIV-1 production in P4P cells. The culture supernatants collected 24 and 48 h after HSV-2 superinfection were used to infect Vero cells. After 48 h of infection, no HIV-1 proviral DNA was detected in the cell pellet, while HSV-2 production was confirmed by the detection of HSV-2 DNA in the supernatant (not shown). Co-infected P4P cells did not produce HIV-1 particles able to infect Vero cells. To check the infectiousness of HIV particles produced in the supernatant of co-infected P4P cells, we tested 24 and 48 h culture supernatants from dually HIV-1 and HSV-2 infected P4P cells on uninfected P4P cells for HIV-1 replication. As expected, these supernatants induced HIV productive infection at 24 hours (not shown), thus confirming that supernatants recovered after co-infection of P4P cells contained infectious HIV-1 particles.
Similar results were observed for both epithelial cell lines with R5-tropic strains HIV Ba-L and HIV JR-CSF (not shown). Thus co-infection of intestinal or endometrial epithelial cells with HIV-1 and HSV-2 did not lead to the production of HIV-1 virions with the ability to infect cells naturally non-permissive for HIV-1.
HIV-1 particles produced from P4P cells expressing HSV-2 gB or gD
In order to avoid the necrosis and apoptosis of epithelial cells consequent to HSV-2 infection, we transfected HIV-1 NDK-infected P4P cells with plasmids coding for gB and gD, which are major glycoproteins involved in fusion of the HSV-2 envelope with the plasma membrane. Culture supernatants obtained 48 h following the transfection were used to infect Vero cells for 24 h. Cell DNA was then extracted, and tested for HIV-1 DNA. Proviral HIV-1 DNA was never detected, suggesting that the expression of gB and gD in HIV-1 infected P4P cells did not modify the phenotype of HIV-1 viral particles such that they could infect Vero cells.
Viral capture assay
In order to assess weteher the colocalization of HSV-2 and HIV glycoproteins can lead to the incorporation of the former into the HIV-1 envelope, we carried out a viral capture assay. Supernatants of HSV-2 and HIV-1 dually infected P4P and CEM cells and of P4P cells infected by HIV-1 then transfected with plasmids coding for gB or gD were first incubated with mouse antibody to gB or gD and biotin-labeled anti-mouse antibody and second with streptavidin microbeads to capture viral particles bearing gB or gD glycoproteins. The positive control capture of HIV-1 particles was assessed by anti-human CD44. Bound viruses were further tested for the detection of HIV-1 RNA and HSV-2 DNA. Capture assays with gB or gD specific antibodies were able to bind HSV-2 virions (Figure 6). No HIV-1 RNA was detected, either from dually infected cells or from gB or gD transfected HIV-1 positive cells, suggesting that HIV-1 particles did not acquire gB or gD glycoproteins during budding (Figure 6).