HIV-1 Infection Is Blocked at an Early Stage in Cells Devoid of Mitochondrial DNA

Human immunodeficiency virus type I (HIV-1) exploits various host cellular pathways for efficient infection. Here we report that the absence of mitochondrial DNA (mtDNA) in ρ0 cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ0 cells is not simply the result of impaired oxidative phosphorylation (OXPHOS) because pharmacological OXPHOS inhibition did not inhibit HIV-1 infection. Analysis of the early steps of virus infection by real-time PCR quantification of stage-specific HIV-1 DNA products in the infected ρ0 and parental cell line have allowed us to conclude that HIV-1 infection in ρ0 cells is blocked at the steps that occur after reverse transcription and prior to nuclear import. Additionally, confocal fluorescence microscope analysis showed that the majority of viral complexes containing HIV-1 p24 co-localize with mitochondria in target cells, suggesting an interaction between the two. Collectively, our data strongly indicate that mitochondria play an important role during early stages of HIV-1 infection, probably through direct association with HIV-1 intracellular complexes.

all HIV-1 DNA synthesized including early RT products (strong stop DNA), late RT products (full length cDNA) and partial RT products. Linearized pWPI plasmids were used as the standard in the measurement of total and late RT products. Integrated HIV-1 DNA was measured by Alu-LTR based real-time nested-PCR procedure [2]. Total cellular DNA isolated from infected HOS cells 40 days post-infection (17% GFP-positive) was used as standard. Integrated HIV-1 DNA in the standard was quantified using the late RT primer set. DNA fragments containing 2LTR junction sequence (U5/U3) or mtDNA (nucleotide position 10620-10710) were cloned into plasmid containing pGEX-3X (Addgene) backbone separately and the recombinant plasmids (p2LTR or pGST-MT) were used as standards in the measurement of 2LTR circles or mtDNA. Cell number variation was measured using real-time PCR for RNase P DNA by using Taqman Copy Number Reference Assay, RNase P (Invitrogen). Cellular DNA isolated from a known number of HOS cells was used as standard in the measurement of cell number variation.

Effect of mitochondrial inhibitor on virus infection. HOS cells were seeded in 12-well
plates at a concentration of 4 X 10 4 /well and incubated overnight at 37°C. Cells were preincubated with various concentrations of different mitochondrial inhibitors (5 µg/ml antimycin A, 10 µg/ml oligomycin and 10 µM carbonyl cyanide 3chlorophenylhydrazone (CCCP) (Sigma) for 6 hours with or without the supplementation of 1 mM sodium pyruvate and 50 µg/ml uridine, and cells were subsequently infected with an equivalent of 10 ng of p24 of HIV-GFP. Two hours after infection, the medium was replaced with fresh medium containing different mitochondrial inhibitors. Infection was assessed 2 days later by FACS analysis as described in the main text.
Repopulation of ρ 0 cells with functional mtDNA. Reintroduction of mtDNA from HEK293T cells into ρ 0 HOS 143B-TKcells was performed essentially as described [3,4]. The use of a TK + cell line, such as HEK293T, as mtDNA donor was required to ensure a proper cybrid selection. HEK 293T cells were seeded in 6-well plates and grown to 70~80% confluency. Cells were enucleated by the treatment with 3 µg/ml actinomycin D for 20 hours and washed with DMEM for 5 min to remove the drug. ρ 0 cells (1X10 6 /well) were added on top of actinomycin D treated HEK293T cells and incubated at 37 °C for 5 hours allowing ρ 0 cells to attach and make contact with HEK293T cells.
Cells were washed three times with DMEM without FBS. One ml of freshly prepared fusion medium (1 g/ml PEG 1450, 20% DMSO in DMEM) was added to the cells and incubated for 2 min at room temperature. The cells were washed with DMEM three times and incubated overnight in complete medium. The cells were detached and reseeded at low concentration (1:20 dilution) in 6-well plates in DMEM supplemented with 10% dialyzed FBS and 100 µg/ml bromodeoxyurine (BrdU), to select against expression of the TK + phenotype of the HEK293T cells, and in the absence of uridine and pyruvate, to select against the respiratory-null unfused ρ 0 cells [3]. Medium was changed every 3 days. Therefore, only the ρ 0 cells that had fused with cytoplasts could survive in this medium, while the ρ 0 cells that had not fused or that had fused with residual intact HEK293T cells, as well as any residual intact HEK293T cells, were eliminated. Colonies  Tables   Table S1. Primers and probes used in this study.

Target DNA Primer or probe
Name and sequence Ref.

Early RT Forward
Early RT Reverse Early RT probe     hours. Mitochondria were labeled with MitoTracker red (Red). Viral complexes were detected with a monoclonal anti-p24 antibody and visualized with Alexa 488-conjugated anti-mouse IgG secondary antibody (Green). Imaging was performed with a laserscanning confocal microscope (LSM510, Carl Zeiss). Cell images were acquired by using the 63X oil objective lens, with zoom factor of 2. Optical slices were taken every 200nm interval along the z-axis covering the whole depth of the cell. Images were analyzed using ImageJ and Zeiss LSM Image Browser. Images (A-I) are representatives of a series of images of one representative infected cell captured at different focal depths along the z-axis.