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. 2001 Aug;75(15):6914-22.
doi: 10.1128/JVI.75.15.6914-6922.2001.

Characterization of a late entry event in the replication cycle of human immunodeficiency virus type 2

McKnight A  1 , D J Griffiths, M Dittmar, P Clapham, E Thomas
Affiliations

Characterization of a late entry event in the replication cycle of human immunodeficiency virus type 2

McKnight A et al. J Virol. 2001 Aug.

Abstract

Certain human cell lines and primary macrophage cultures are restricted to infection by some primary isolates of human immunodeficiency virus type 2 (HIV-2), although early steps of the viral life cycle such as fusion at the plasma membrane and reverse transcription are fully supported. The late postintegration events, transcription, translation, assembly, budding, and maturation into infectious virions are functional in restrictive cells. Apart from primary macrophages, the restrictive cell types are actively dividing, and nuclear import of preintegration complexes (PICs) is not required for infection. We therefore postulate that the PICs are trapped in a cellular compartment, preventing subsequent steps in the replication cycle that lead to integration of the provirus. To test this we showed that HIV-2 particles pseudotyped with vesicular stomatitis virus envelope G protein, which delivers HIV into an endocytic compartment, could overcome the block to infection. We suggest that delivery of the viral core into an appropriate cellular compartment is a critical step during the entry process of HIV.

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Figures

FIG. 1
FIG. 1
PrCBL-23 but not CBL-23 is restricted for infection on HeLa/CD4 cells, HOS/CD4/CXCR4 cells and primary macrophages. (A) The infectious titer of prCBL-23 (solid bars) compared to TCLA CBL-23 (shaded bars) is shown for HeLa/CD4, HOS/CD4/CXCR4, and U87/CD4/CXCR4 cells. Virus-infected cells were fixed, and stained, and foci of infection were counted and calculated. CBL-23 can efficiently infect all three cell lines tested, whereas infection of HeLa/CD4 and HOS/CD4/CXCR4 cells is restricted for prCBL-23, even though it can efficiently infect U87/CD4/CXCR4 cells. (B) Comparative time course of infection by prCBL-23 (dotted line) and CBL-23 (solid line) on the T-cell line C8166, primary PBMCs, and primary macrophages. Equal quantities of infectious units (100 FFU, estimated on U87/CD4/CXCR4 cells) of both viruses were inoculated into cells, and the release of infectious virus was measured by RT activity and expressed as picograms per milliliter. The kinetics of infection shows that prCBL-23 can efficiently infect both C8166 cells and PBMCs but not macrophages. The kinetics of infection on PBMCs is, however, delayed (2 to 3 days) for prCBL-23 compared to CBL-23.
FIG. 2
FIG. 2
DOTAP overcomes the restriction to infection by prCBL-23 in HeLa/CD4 and HOS/CD4/CXCR4 cells. (A) HeLa/CD4 cells were challenged either with untreated virus (left panels) or with virus treated with 20 μg of DOTAP/ml (right panels). Infected cells are blue after immunostaining. (B) HOS/CD4/CXCR4 cells challenged either with untreated virus (left panels) or with DOTAP-treated virus (right panels). (C) The enhancement of infection was estimated for DOTAP-treated virus plated on HeLa/CD4 cells (left) and HOS/CD4/CXCR4 cells (right). Shaded bars, untreated virus; solid bars, DOTAP-treated virus. Focus-forming units per milliliter were estimated after immunostaining
FIG. 3
FIG. 3
The envelope of prCBL-23 can induce cell-to-cell fusion. The figure shows cell-to-cell fusion of prCBL-23 and CBL-23 with HeLa/CD4 and HOS/CD4/CXCR4 cells. PrCBL-23- or CBL-23-infected C8166 cells were cocultivated overnight with _target cells. Adherent cells were then fixed and stained with methylene blue. (Top and bottom left) Coculture of uninfected C8166 cells with HOS/CD4/CXCR4 and HeLa/CD4 cells, respectively. (Middle and right) Cocultivation of the same _target cells with either prCBL-23- or CBL-23-infected C8166 cells, respectively, results in multinucleated giant cells or syncytia.
FIG. 4
FIG. 4
The envelope of prCBL-23 confers entry into, and infection of, HeLa/CD4 cells. HeLa/CD4 and U87/CD4/CXCR4 cells were challenged with ROD or a pseudotype virus, prCBL-23/ROD, that contains mixed particles with the structural and envelope glycoproteins of both ROD and prCBL-23. Cells were challenged with the pseudotype in the presence of an anti-envelope neutralizing antibody which specifically and potently neutralizes ROD envelope-mediated infectivity but not that of prCBL-23 (40). The infectivity of the ROD virus (nonpseudotyped) is neutralized on both cell types by at least two log units of infectivity (A). In contrast, the pseudotype virus ROD/prCBL-23 was rescued by prCBL-23 glycoproteins and is not neutralized significantly on either cell type (B). The infectivity of ROD/prCBL-23 on HeLa/CD4 cells represents infectivity mediated by the prCBL-23 envelope.
FIG. 5
FIG. 5
PCR analysis of reverse transcripts. (A) LTR-gag PCR products from cell lines infected with CBL-23 and prCBL23 (input was 100 FFU as determined on U87/CD4/CXCR4 cells). Cells were harvested 24 h after infection. For each cell line, equivalent levels of LTR-gag were produced for CBL-23 and prCBL-23. Lanes: 1, 10,000 cells; 2, 3,333 cells; 3, 1,000 cells; 4, 333 cells; 5, 100 cells, 6, 33 cells. (B) (Top) LTR-gag products amplified from 104 macrophages (MAC) or PBMCs infected with CBL-23 (C) or prCBL-23 (Pr) at various times postinfection. Macrophages infected with prCBL-23 have late transcripts after 24 h, but these do not persist to later time points. (Bottom) Limiting-dilution PCR analysis for the single-copy human genomic sequence ERV-3 on samples taken at 48 h. All samples have comparable DNA levels.
FIG. 5
FIG. 5
PCR analysis of reverse transcripts. (A) LTR-gag PCR products from cell lines infected with CBL-23 and prCBL23 (input was 100 FFU as determined on U87/CD4/CXCR4 cells). Cells were harvested 24 h after infection. For each cell line, equivalent levels of LTR-gag were produced for CBL-23 and prCBL-23. Lanes: 1, 10,000 cells; 2, 3,333 cells; 3, 1,000 cells; 4, 333 cells; 5, 100 cells, 6, 33 cells. (B) (Top) LTR-gag products amplified from 104 macrophages (MAC) or PBMCs infected with CBL-23 (C) or prCBL-23 (Pr) at various times postinfection. Macrophages infected with prCBL-23 have late transcripts after 24 h, but these do not persist to later time points. (Bottom) Limiting-dilution PCR analysis for the single-copy human genomic sequence ERV-3 on samples taken at 48 h. All samples have comparable DNA levels.
FIG. 6
FIG. 6
VSV-G envelope can overcome the block to infection on restrictive cell types. Infectious titers (measured in focus-forming units per milliliter) of CBL-23, prCBL-23, and VSV-G(prCBL-23) were measured on U87/CD4/CXCR4, HeLa/CD4, and HOS/CD4/CXCR4 cells. The VSV-G envelope rescues prCBL-23 infection of Hela/CD4 and HOS/CD4/CXCR4 cells.
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