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Defective HIV-1 Proviruses Can Be Transcribed Upon Activation
Ya-Chi Ho1, Ross Pollack1, Patrick Yong1, Robert F. Siliciano1
1 Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Background: HIV-1 persists in the latent reservoir, primarily resting memory CD4+ T cells, as integrated proviruses. The majority of these proviruses are defective, containing large internal deletions or APOBEC-mediated G-to-A hypermutations. However, we previously found that even if the HIV-1 genome contains lethal mutations, the LTR promoter may remain intact, indicating that HIV-1 RNA may still be transcribed. The transcription of HIV-1 RNAs from defective proviruses may complicate the measurement of the size of the latent reservoir using latency reversing agents during the shock-and-kill strategy, as measurement of the defective proviral RNA does not indicate the reactivation of the clinically significant replication-competent proviruses. Further, whether the cells harboring defective proviruses would expand upon reactivation, or would be eliminated by cytolytic T cells (CTLs), remains unknown.
Methods: Resting CD4+ T cells from aviremic patients under suppressive antiretroviral therapy are activated with CD3/CD28 costimulation under enfurvitide to prevent new rounds of in vitro infection. Autologous CTLs were stimulated with Group M Consensus Gag peptide mixture and IL-2. To examine whether cells containing intact or defective HIV-1 can be eliminated by CTLs, we co-cultured pre-stimulated autologous CTLs with activated CD4+ T cells. Cell-associated RNA and proviral DNA from cells which are resting, activated, and CTL co-cultured was subjected to quantitative PCR and deep-sequencing of the Gag region to examine the start codon of Gag and two tryptophan residues, which are hotspots APOBEC-mediated hypermutations. CTLs were removed by magnetic bead depletion from the CTL-CD4 coculture before qPCR for normalization to CD4 cell count.
Results: We found a significant proportion of the HIV-1 RNA in activated patient CD4+ T cells contains lethal mutations. The amount of defective proviruses increased over the course of activation, indicating expansion of cells containing defective proviruses upon stimulation. The percentage of defective proviruses increased, implying the effect of viral cytopathic effects by reactivated intact proviruses. The amount of HIV-1 proviruses, both intact and defective, decreased after addition of CTLs in some patients, indicating possible elimination by CTLs.
Conclusions: Defective HIV-1 proviruses may be transcribed during latency reversal. Cells containing defective HIV-1 proviruses may expand under T cell activation.