HIV-1 Vpr-induced DNA damage activates NF-?B through ATM-NEMO independent of cell cycle arrest
Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through DNA damage, cell cycle arrest, the degradation of host proteins, and both the activation and repression of DDR signaling. Vpr also alters host and viral transcription; however, the connection between Vpr-mediated DDR modulation and transcriptional activation remains unclear. Here, we determined the cellular consequences of Vpr-induced DNA damage using Vpr mutants that allow us to separate the ability of Vpr to induce DNA damage from cell cycle arrest and other DDR phenotypes including host protein degradation and repression of DDR. RNA-sequencing of cells expressing Vpr or Vpr mutants identified that Vpr alters cellular transcription through mechanisms both dependent and independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), Vpr-induced DNA damage activates the ATM-NEMO pathway and alters cellular transcription via NF-?B/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-?B transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-?B transcription, suggesting that engagement of the DDR and transcriptional reprogramming can occur during early and late stages of viral replication. Together, our data identifies a mechanism by which Vpr activates NF-?B through DNA damage and the ATM-NEMO pathway, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral transcription and replication. Overall design: To determine if Vpr WT and mutants alter the cellular transcriptome, we performed RNA-seq on U2OS cells infected with recombinant adeno-associated virus (rAAV) expressing either Vpr WT or Vpr mutants S79A, H71R and Q65R. U2OS cells treated with empty vector rAAV were used as negative vehicle control, and untreated U2OS cells served as negative control. Total RNA was collected at 36 hours post infection. Experiment was done with technical triplicates. Using the statistical significance of Log2 (1.25), p < 0.01, and FDR < 4.50E-05, We found that Vpr WT, H71R and S79A alters cellular transcription compared to untreated cells. Furthermore, Vpr Q65R and empty vector rAAV treated cells did not alter transcription compared to untreated cells.