HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

Abstract

Background: Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1.

Methods: We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD.

Results: We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells.

Conclusion: Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2.

Figure 1

Co-staining of HSV-2 gB or gD with HIV-1 gp160. HIV-1 infected P4P cells were either superinfected by HSV-2 (A-F) or transfected by a plasmid coding for gB (G-I) or gD (J-L). Cells were stained by immunofluorescence for the co-detection of gp160 and gB (A-C, G-I) or gp160 and gD (D-F, J-L), using human polyclonal anti-gp160 followed by FITC-labelled F(ab')2 rabbit anti-human IgG giving a green staining for HIV-1 gp160 and mouse Mabs against gB or gD followed by a TRITC-labelled rabbit anti-mouse IgG giving a red staining for HSV-2 gB or HSV-2 gD.

Figure 2

HIV-1 replication in HIV-1 infecetd CEM cells co-infected by HSV-1 or HSV-2. HIV-1 RNA has been quantified in the supernatant of CEM cells every 24 h up to 72 h following superinfection by HSV-1 (A) or HSV-2 (B) at an MOI of 1 (dark grey), 0.1 (light grey) and 0.01 (white). Cells not superinfected are represented in black. Values are expressed in log₁₀ copies/ml. Data are representative of three independent experiments.

Figure 3

HSV-1 and HSV-2 replication in CEM cells co-infected by HIV-1. HSV-1 (A) and HSV-2 (B) viral load, expressed in log₁₀ copies/ml, has been determined in the supernatant of CEM cells infected by HSV-1 or HSV-2 at an MOI of 1 (dark grey), 0.1 (light grey) and 0.01 (white) and in HIV positive CEM cells superinfected by HSV-1 or HSV-2 at an MOI of 1 (dotted dark grey), 0.1 (dotted light grey) and 0.01 (dotted white) for up to 72 h.

Figure 4

HIV-1 expression in dually HIV-1 and HSV-2 infected HT29 cells. HIV-1 production in infected HT29 cells 24 h and 48 h after HSV-2 superinfection at the MOI of 1 (dark grey), 0.1 (light grey) and 0.01 (white). Values, expressed in pg/ml of p24 Ag, are the mean of three independent experiments. Control HIV-1 infected HT29 cells not superinfected by HSV-2 are represented in black.

Figure 5

HIV-1 expression in dually HIV-1 and HSV-2 infected P4P cells. HIV-1 production in HIV-1 infected P4P cells 24 h and 48 h after HSV-2 superinfection at the MOI of 1 (dark grey), 0.1 (light grey) and 0.01 (white). Values of HIV-1 RNA loads, expressed in log₁₀ copies/ml, are the mean of three independent experiments. Control HIV-1 infected HT29 cells not superinfected by HSV-2 are represented in black.

Figure 6

Viral capture assay. HIV-1 and HSV-2 capture by antibodies targeted against cell surface CD44 antigen or HSV-2 gB or gD in HIV-1- and HSV-2 dually infected P4P cells (A), CEM cells (B) and HIV-1-infected P4P cells transfected by gB or gD (C). Levels of captured virus were determined by HIV-1 RNA (open square) and HSV-2 DNA (bold square) quantification by real time PCR and compared to capture by a negative control antibody (biotin labeled anti-mouse) to derive fold-over background values. Data are representative of three independent experiments.