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. 2001 Mar;75(5):2288-300.
doi: 10.1128/JVI.75.5.2288-2300.2001.

Use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and T tropism: evidence for distinct levels of restriction in primary macrophages and a T-cell line

S S Kim  1 , X J You, M E Harmon, J Overbaugh, H Fan
Affiliations

Use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and T tropism: evidence for distinct levels of restriction in primary macrophages and a T-cell line

S S Kim et al. J Virol. 2001 Mar.

Abstract

Cell tropism of human and simian immunodeficiency viruses (HIV and SIV, respectively) is governed in part by interactions between the viral envelope protein and the cellular receptors. However, there is evidence that envelope-host cell interactions also affect postentry steps in viral replication. We used a helper-free replication-defective SIV macaque (SIVmac)-based retroviral vector carrying the enhanced jellyfish green fluorescent protein inserted into the nef region (V1EGFP) to examine SIV tropism in a single cycle of infection. Vector stocks containing envelope proteins from three different SIVmac clones, namely, SIVmac239 (T-lymphocyte tropic [T-tropic]), SIVmac316 (macrophage tropic [M-tropic]), and SIVmac1A11 (dualtropic), were tested. SIVmac239 replicates efficiently in many human T-cell lines, but it does not efficiently infect primary rhesus macrophages. Conversely, SIVmac316 efficiently infects primary macrophages, but it does not replicate in Molt4-Clone8 (M4C8) T cells. SIVmac1A11 replicates efficiently in both cell types. When primary macrophages were infected with V1EGFP pseudotyped by SIVmac316 or SIVmac1A11 envelopes, the infection was substantially (ca. 200- to 300-fold) more efficient than for the SIVmac239 pseudotype. Thus, in primary macrophages, a major component of M versus T tropism involves relatively early events in the infection cycle. Quantitative PCR studies indicated that synthesis and transport of vector DNA into the nucleus were similar for macrophages infected with the clone 239 and 316 pseudotypes, suggesting that the restriction for SIVmac239 infection is after reverse transcription and nuclear import of viral DNA. When the same vector pseudotypes were used to infect M4C8 cells, they all showed approximately equivalent infectivities, even though replication-competent SIVmac316 does not continue to replicate in these cells. Therefore, in M4C8 cells, restriction involves a late step in the infection cycle (after proviral integration and expression). Thus, depending on the cell type infected, envelope-dependent cell interactions that govern SIV M and T tropism may involve different steps in infection.

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Figures

FIG. 1
FIG. 1
SIV vector and packaging plasmids. Schematic diagrams of the SIVmac239-based vector V1EGFP (A), the packaging plasmid pUpSVOΔΨ (B), and the envelope expression plasmids (C) are shown. (A) V1EGFP is replication defective due to a deletion in env and two consecutive stop codons at the beginning of gag. (B) pUpSVOΔΨ contains a deletion in env and the packaging sequence (Ψ) and contains a heterologous murine leukemia virus 3′ long terminal repeat (LTR). (C) The envelope expression plasmids all contain the cytomegalovirus immediate-early promoter driving the expression of env, rev, and nef; different plasmids contain env sequences from different SIVmac strains.
FIG. 2
FIG. 2
Infection with V1EGFP vector. Cultures of CMMT-CD4 cells or primary rhesus macrophages were infected with undiluted stocks of V1EGFP pseudotyped with SIVmac1A11 envelope. The cultures were then examined by fluorescence microscopy with a green filter. (A) Infected CMMT-CD4 cells 4 days postinfection. (B) Uninfected CMMT-CD4 cells. (C) Infected rhesus macrophages 4 days postinfection. (D) CMMT-CD4 cells infected in the presence of the reverse transcriptase inhibitor PMPA.
FIG. 3
FIG. 3
Fluorescence intensity of V1EGFP-infected cells. Cultures of CMMT-CD4 cells (5 × 104) were infected with V1EGFP pseudotyped with different SIVmac envelopes, in the presence or absence of PMPA. The cultures were then harvested at 4 days postinfection, fixed, and analyzed for green fluorescence by flow cytometry. The x axis shows log fluorescence intensity and the y axis shows cell number; 10,000 cells were analyzed in each case. (A and B) Cells infected with a SIVmac239 pseudotype (1.6 × 104 GFU); (C and D) cells infected with a SIVmac1A11 pseudotype (4 × 103 GFU); (E and F) cells infected with a SIVmac316 pseudotype (8 × 103 GFU); (G and H), uninfected cells. (B, D, F, and H), cultures infected in the presence of 100 μM PMPA. The vector-infected cells are evident as the peak with a mean fluorescence of ca. 200 to 300. Note that the fluorescence values for the cultures infected with the different pseudotypes were approximately the same.
FIG. 4
FIG. 4
Fluorescence intensity of V1EGFP-infected macrophages. Cultures of PBMC-derived macrophages were infected with V1EGFP pseudotyped with different SIVmac envelopes (4 × 104 GFU on CMMT-CD4 cells per 105 cells). The cultures were then harvested at 4 days postinfection, fixed, and analyzed for green fluorescence by flow cytometry. The x axis shows log fluorescence intensity, and the y axis shows cell number; 5,000 cells were analyzed in each case. (A) Cells infected with a SIVmac316 pseudotype; (B) cells infected with a SIVmac1A11 pseudotype; (C) cells infected with a SIVmac239 pseudotype. The fluorescence values for the cultures infected with the different pseudotypes were approximately the same. The geometric mean intensities (in arbitrary fluorescence units) in the M1 region were as follows: 601 for the SIVmac239 pseudotype, 1,212 for the SIVmac1A11 pseudotype, and 1,265 for the SIVmac316 pseudotype.
FIG. 5
FIG. 5
Quantification of vector DNA in infected macrophages. (A) PBMC-derived rhesus macrophages (105) were infected with DNase-treated V1EGFP pseudotyped with either SIVmac239 or SIVmac316 Env protein, at a multiplicity of 0.025 GFU (titered on CMMT-CD4 cells) per cell. At 24 h after infection, nuclei were prepared and DNA was extracted. Equal samples of nuclear DNA were tested for the presence of vector DNA by real-time PCR in a TaqMan thermal cycler, using the SIV-specific PCR primers and probe described in Materials and Methods. Quantification (A260) of the total nuclear DNAs prior to real-time PCR indicated equivalent efficiencies of recovery. The relative fluorescence signal for each PCR cycle is shown for each DNA sample. Duplicate amplifications were performed for each DNA. As a control, nuclear DNA from uninfected macrophages was analyzed in parallel. Fluorescence values below 10−2 were not significant. (B) In a second experiment, rhesus macrophages were infected with SIVmac239 and SIVmac316 pseudotypes of V1EGFP, and nuclear DNA was quantified as described for panel A. In addition, macrophages were infected with the SIVmac239 pseudotype of V1EGFP in the presence of 200 μM PMPA and analyzed in parallel. Results from duplicate real-time PCR assays are shown.
FIG. 6
FIG. 6
Timing of reverse transcription in macrophages infected with vector pseudotypes (monkey 25980). A total of 4 × 105 macrophages in 12-well plates were infected with 500 μl of diluted V1EGFP stocks pseudotyped with different SIVmac envelopes as described in Materials and Methods. Each well infected with the SIVmac239 pseudotype received 6.3 × 103 GFU, each well infected with the SIVmac1A11 pseudotype received 1.6 × 103 GFU, and each well infected with the SIVmac316 pseudotype received 1.7 × 103 GFU. A 100 μM concentration of PMPA was added at the time points indicated. All cultures were scored for infected cells at 4 days postinfection. The levels of infection are plotted as percent infection without PMPA.
FIG. 7
FIG. 7
Replication of SIVmac viruses in Molt4-Clone8 cells. A total of 5 × 105 Molt4-Clone8 cells were infected at a multiplicity of infection of 0.002 with different SIVmac viruses as described in Materials and Methods. p27 SIV gag antigen in culture supernatants was measured at different times and is plotted versus days postinfection.
FIG. 8
FIG. 8
Timing of reverse transcription in Molt4-Clone8 cells infected with vector pseudotypes. A total of 106 Molt4-Clone8 cells in six-well plates were infected with 105 GFU of V1EGFP pseudotyped with different SIVmac envelopes. A 100 μM concentration of PMPA was added at the times indicated, and the numbers of GFP-positive cells were measured at 4 days postinfection by flow cytometry. Infection levels are shown as percentage of GFP-positive cells in the absence of PMPA.
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