Background
BACKGROUND
Persistence of the HIV reservoir during antiretroviral therapy is a major obstacle to curing the infection. This reservoir can be established in the hyperacute stage of the infection, and lymphoid tissue analysis of this stage revealed HIV infection of resting T cells. Given that the HIV reservoir resides in resting CD4 T cells, we investigated whether direct infection of resting CD4 T cells could be involved in its establishment.
Methods
METHODS
We infected primary resting CD4 T cells with a CCR5-tropic replication-competent GFP reporter virus. We co-measured GFP expression with activation and proliferation markers by flow cytometry over five days. We performed bulk and single-cell RNAseq of sorted GFP+ cells to measure host and virus mRNAs. We bioinformatically analyzed cellular dNTP pathways for HIV reverse transcription and read-through HIV transcripts for HIV integration. Lastly, we cross-validated our findings with peripheral and lymphoid tissue samples from people living with HIV (PLWH).
Results
RESULTS
We detected resting GFP+ cells 3 to 5 days after infection. These GFP+ cells lacked CD69, CD25, HLA-DR, and CD154 expression. They also did not express the proliferation markers Ki-67 and phospho-H3.3. Transcriptome analysis of resting GFP+ cells revealed that infected cells expressed a cell-cycle independent pathway for dNTP synthesis. This pathway lacked expression of the genes required to convert deoxyuracil (dU) to thymidine (dT), which resulted in incorporation of dU into the provirus. We observed that dU-containing (dU+) proviruses were integrated into the host genome. We designed and ran a qPCR assay that differentiated between proviruses with dU or dT on samples from PLWH. dU+ proviruses and dT+ proviruses were detected in different CD4 T cell populations in all donors tested. Finally, after performing viral outgrowth assays, we found that cell subsets with dU+ proviruses had replication-competent virus.
Conclusions
CONCLUSIONS
We conclude that HIV can directly infect primary resting CD4 T cells and establish the reservoir in these cells. A key feature of HIV infection of resting cells is dU incorporation during reverse transcription due to the absence of enzymes required for dT synthesis. Therefore, the HIV reservoir comprises integrated, replication-competent, deoxyuracil-containing proviruses.
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