PMC

A schematic model summarizing the two pathways of HIV-1 entry into cells, including CD4- and chemokine-receptor-dependent fusion at the plasma membrane and receptor-independent endocytosis. HIV-1 virions entering by fusion at the plasma membrane support subsequent steps in the retroviral life cycle leading to HIV-1 replication and viral spread, while virions entering by endocytosis in general do not lead to productive infection. The nef gene product, acting either directly or indirectly, significantly enhances cytoplasmic virion entry occurring by fusion.

Analysis of infectivity of HIV viruses containing wild-type or mutant nef genes. Viral replication was measured in Jurkat T cells infected with the indicated viruses. Levels of p24 Gag released into the culture medium were measured 3 days after infection. Values are representative of three independent experiments performed in duplicate. Standard deviation was less than 15% for all measurements.

Effect of nef mutations on cytosolic p24^gag entry into HeLa-CD4 cells. HeLa-CD4 cells were incubated with viruses containing wt nef, a nef deletion (Δnef), or various alanine substitution mutants of Nef for 1 h at 37°C and fractionated as described in the legend to Fig. . Histograms present the percentage of p24^gag present in the cytosolic fraction of these cultures. Note that mutation of nef either in its central proline helix or at two sites implicated in CD4 downregulation significantly impairs the cytosolic appearance of p24^gag.

HIV efficiently enters HeLa cells lacking CD4 and localizes in endosomes. (A to D) Green-fluorescing HIV-1 virions were produced by cotransfection of 293T cells with expression vectors encoding GFP-Vpr and either pNL4-3, containing the wt nef gene, or pNL4-3Δnef, containing a nef deletion mutant. HeLa-CD4 or HeLa cells grown on coverslips were incubated with these fluorescent virions (200 ng of p24^gag) in 80 μl for 20 min at 37°C. The cells were processed for fluorescence microscopy as described in Materials and Methods. Note significant binding and entry of green-fluorescing virions into HeLa cells. (E to J) In addition to exposure to wild-type fluorescent HIV virions, HeLa cells were incubated with TAMRA-Tf to fluorescently label endosomes and with LysoTracker Red to fluorescently label lysosomes. Overlay of the combined fluorescent signals is shown in panels G and J, demonstrating significant colocalization of the GFP-Vpr-labeled viral particles with TAMRA-Tf.

Nef enhances HIV-1 entry into the cytosol of HeLa-CD4 and Jurkat T cells. HeLa-CD4 cells or Jurkat T cells were infected with HIV viruses containing the wt nef gene or the Δnef mutation or viruses lacking the HIV envelope gene (Δenv). Additionally, the wt nef and or Δnef viruses were tested after pseudotyping with the VSV-G envelope (VSV env) (500 ng of p24^gag). After a 1-h incubation of cells and viruses at 37°C, cytosolic and endosomal fractions were prepared as described in Materials and Methods. The level of p24^gag present in each of these fractions was measured by ELISA. Values correspond to the percentages of p24^gag in the cytosol relative to the total intracellular p24 Gag after substraction of the background values measured with the Δenv mutant. Data are representative of three to six experiments performed with three independent virus preparations. Cytosolic delivery averaged 0.1% of total p24^gag input for HIV-1 and 0.6% for HIV pseudotyped with the VSV-G envelope.

Analysis of binding, entry, and replication of CXCR4- and CCR5-tropic HIV containing wt nef or Δnef in HeLa-CD4 and HeLa cells. (A) Similar levels of HIV-1 Nef wt and ΔNef bind to and enter HeLa-CD4 and HeLa cells at 4 h. Confluent cells were inoculated with HIV-1 pNL4-3 wt X4 and ΔNef X4 (50 ng of p24) and incubated at either 4 or 37°C, as indicated. After 4 h, the cells were washed with PBS and either trypsinized (+T) or washed with PBS (−T). Cells were then washed twice with PBS, and cell lysates were prepared. Levels of p24^gag in the cell lysates were measured by ELISA. Data represent averages of three independent infections performed in duplicate. Note comparable entry of both HIV wt nef and HIV Δnef virions into HeLa-CD4 cells and persistent entry of these viruses into HeLa cells lacking CD4. (B) Entry of CXCR4-tropic or CCR5-tropic strains of HIV-1 containing wt nef or Δnef into HeLa-CD4 cells. Assays were performed as described above except that the cells were incubated with virions for 30 min at 37°C. Note effective entry of R5-tropic HIV into HeLa-CD4 cells lacking CCR5. (C) Analysis of HIV replication in HeLa-CD4 and HeLa cells. The viruses and cells described for panels A and B were used to study viral replication. Samples of the culture supernatant were analyzed for p24^gag content on days 2 to 7 of short-term cultures. Data shown are from a representative experiment performed three times with comparable results. Note that effective viral replication occurred only when CXCR4-tropic HIV containing the wt nef gene was cultured with HeLa-CD4 cells.