Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells

Abstract

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.

Fig. 1.

Identification of the minimal Vpx packaging motif in SIVmac p6 by transfer into HIV-1 p6. (A) Alignment of the NL4-3 and SIVmac₂₃₉ p6 amino acid sequences. The two proposed α-helices (α1 and α2) are shaded, and the PTAPP late domain and TSG101 binding site are boldface and underlined. Binding sites for ALIX and Vpx are also boldface and underlined. Below the alignment are diagrams of the HIV-1/SIVmac p6 chimeras. Open rectangle, HIV-1 sequence; filled rectangle, SIV sequence. The SIV sequence is inserted at position 14 of HIV-1 p6, except in chimera 17-23(a), where the Vpx binding motif is displaced to position 21 in order not to alter the ¹⁵FRFG¹⁸ Vpr packaging motif. (B) Immunoblot analysis shows packaging of SIVmac₂₃₉ Vpx in chimeras containing the Vpx packaging motif. 293T cells were cotransfected with pNL4-3 containing wild-type (WT) or chimeric p6 and either pcVpx.myc or an empty vector. Two days later, cell lysates and virions were pelleted by ultracentrifugation and were analyzed by immunoblotting. The immunoblot was probed with an antibody to myc-tagged Vpx, HIV-1 p24 CA, or tubulin. Transfection with pcVpx-myc alone was included in order to rule out nonspecific release of Vpx. (C) HIV-1 p6 chimeras that map amino acids required for Vpx packaging. Virions were prepared by transfection and were analyzed by immunoblotting as for panel B. (D) Effects of p6 mutations on packaging of HIV-1 Vpr. 293T cells were cotransfected with wild-type or chimeric pNL4-3 p6 and either pcVpr.myc or an empty vector. The cell lysate and virions were analyzed by immunoblotting as for panel B.

Fig. 2.

Relative contributions of the two proposed Vpr packaging motifs of HIV-1. (A) Sequences of the two reported Vpr packaging motifs of HIV-1 p6. The two motifs are underlined and boldface. (B) Immunoblot analysis of Vpr packaged by virions with mutations in motif 1. Virions were generated by transfection of 293T cells with pNL4-3 containing wild-type (WT) or mutant p6 and either a pcVpr.myc expression vector or an empty vector. The virions were pelleted from the culture supernatants by ultracentrifugation and were analyzed on an immunoblot probed with an antibody to myc-tagged Vpr, HIV-1 CA p24, or tubulin. (C) Immunoblot analysis of Vpr packaged by virions with mutations in motif 2. Mutant virions were prepared as for panel B. In the M1A mutant, F15, R16, F17, and G18 in motif 1 are mutated to alanine. In the M2Aa mutant, L35, L38, L41, and L44 in motif 2 are mutated to alanine. M2Ab is M2Aa with the additional mutations of S40, S43, and F45 to alanine. “M1 and 2a” is the combination of M1A and M2Aa. “M1 and 2b” is the combination of M1A and M2Ab.

Fig. 3.

Chimeric HIV-1 containing the Vpx packaging motif has an enhanced ability to infect MDDC and MDM. (A) Expression and packaging of Vpx provided in trans. Luciferase reporter viruses were generated by cotransfection of 293T cells with a wild-type (WT) or p6 chimeric (SIVp6 17-26) proviral reporter virus plasmid (18 μg) and increasing amounts of pcVpx.myc (0.3 μg, 0.6 μg, 3.0 μg, and 6.0 μg), with the total mass of DNA held constant by the addition of the pcDNA plasmid. The cell lysate and the resulting virions were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, HIV-1 CA p24, or tubulin. (B) Effects of Vpx on infection of MDDC and MDM. MDDC (top) and MDM (bottom) were infected with VSV-G-pseudotyped luciferase reporter viruses at 300,000 cps based on normalization of infectivity on 293T cells, an amount corresponding to an average MOI of 0.3. After 4 days, the cultures were harvested, and the luciferase activity was determined. The data are displayed as fold enhancement of luciferase activity (calculated as the luciferase activity of the virus containing Vpx divided by that of the virus lacking Vpx). Error bars indicate the standard deviations of triplicate measurements. Results from three MDDC and two MDM donors are shown. (C) p24 analysis of MDDC and MDM infection by HIV-1 containing Vpx. MDDC (top) and MDM (bottom) were infected with a luciferase reporter virus at 20 ng p24, corresponding to an average MOI of 1.1. Three days later, the cells were collected, and intracellular p24-FITC levels were determined by flow cytometry.

Fig. 4.

Comparison of SIVmac Vpx, SIVagm Vpr, and HIV-2_rod Vpx. Luciferase reporter viruses were generated by cotransfection of 293T cells with wild-type (WT) or p6 chimeric HIV-1 and an expression vector for SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, or SIVagm Vpr. (A) Expression and packaging of codon-optimized SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, and SIVagm Vpr. Cell lysates and pelleted virions were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, myc-tagged Vpr, HIV-1 p24 CA, or tubulin. (B) Effects of HIV-1 packaging of SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, or SIVagm Vpr on MDDC and MDM infection. MDDC and MDM were infected with the indicated virus at 300,000 cps based on normalization of luciferase activity on 293T cells, corresponding to an average MOI of 0.3. Four days postinfection, luciferase activity was determined. The data are displayed as fold enhancement of luciferase activity (luciferase activity of the virus containing Vpx divided by that of the virus lacking Vpx). Error bars indicate the standard deviations of triplicate measurements. Results for three MDDC donors and three MDM donors are shown.

Fig. 5.

A CCR5-using chimeric virus with Vpx in cis replicates more efficiently in MDDC and MDM than wild-type virus. Wild-type (WT) and p6 chimeric NL.Ba-L viruses that express Vpx in cis were generated by transfection of 293T cells. (A) cis viruses encoding SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, and SIVagm Vpr produce the accessory proteins. 293T cells were transfected with proviral plasmid DNA and, 5 h prior to harvest, were treated with 20 μM MG132. Cell lysates were prepared and analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, myc-tagged Vpr, or tubulin. (B) The HIV-1 p6 chimeric virus with SIVmac₂₃₉ Vpx in cis packages Vpx. Wild-type and p6 chimeric pNL.Ba-L viruses encoding SIVmac₂₃₉ Vpx virions were pelleted from the culture supernatants of 10 culture dishes (10 times as many dishes than were used in the analysis of the trans virus). Pelleted virions and cell lysates were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, HIV-1 CA p24, or tubulin. For the p24 immunoblot analysis, 1/10 as much virus lysate was used as for the Vpx immunoblot analysis. (C) Vpx packaged by the cis p6 chimeric virus results in the generation of more reverse transcripts (RT). MDM were infected with 5 ng wild-type or cis p6 chimeric virus, corresponding to an average MOI of 0.04. After 24 and 48 h, DNA was prepared, and the reverse transcripts were quantified by qRT-PCR using primers specific for the late products and 2-LTR circles. Early products were not analyzed, because these were found to be present in the virions prior to infection. To control for plasmid contamination and intravirion reverse transcripts, AZT (25 μM) was added to the sample infected with the p6 chimeric virus encoding SIVmac Vpx (SIVp6: vpx⁺). (D) The cis p6 chimeric virus that encodes SIVmac₂₃₉ Vpx is more active than those encoding HIV-2_rod Vpx or SIVagm Vpr. MDDC were infected with 50 ng p24, corresponding to an average MOI of 0.5. After 3 days, the cells were collected, stained for intracellular p24, and analyzed by flow cytometry. AZT (25 μM) was used to control for contamination with the input virus. Nelfinavir (Nel) (3 μM) was used to limit replication to a single cycle. Results from two donors are shown.

Fig. 6.

Infection of MDDC with a Vpx-containing virus stimulates an innate immune response. MDDC from two healthy donors were infected with the p6 chimeric virus complemented in trans with Vpx. The virus was used at 50 ng p24, which corresponded to an average MOI of 2.7. AZT (25 μM) or raltegravir (Ral) (10 μM) was added to the indicated samples. The cultures were harvested 24 and 48 h postinfection, and IFN-β mRNA was quantified by qRT-PCR. The data are presented relative to the levels of G6PDH mRNA amplified in parallel.

Fig. 7.

Exposure of MDDC to a Vpx-containing virus allows for efficient transfer of the virus to T cells. (A) MDDC were infected with NL.Ba-L wild-type or p6 chimeric virus containing vpx in cis, with or without additional Vpx complementation in trans. The virus was added at 50 ng p24, corresponding to an average MOI of 0.24. After 6 h, free virus was removed, and after 48 h, CD3/CD28-activated autologous CD4 T cells were added. Levels of the p24 supernatant were measured over 10 days. Results are representative of MDDC and CD4 T cells from two donors. (B) MDDC were infected as for panel A but with the virus at 25 ng p24, corresponding an average MOI of 0.3. After 6 h, free virus was removed. After 48 h, medium or CD3/CD28-activated autologous T cells were added. In parallel, T cells alone were infected as for panel A, and after 6 h, free virus was removed. Levels of the p24 supernatant were measured over 14 days. The results shown are representative of MDDC and CD4⁺ T cells from two donors. (C) MDDC and T cells were infected as for panel B, and cells were collected at day 3 postcoculture. The cells were incubated with APC-conjugated anti-CD11c and FITC-conjugated anti-p24 and were analyzed by flow cytometry. The cell populations were gated on CD11c and were then evaluated for intracellular p24. The results shown are representative of three donors tested.