Figure 3. TET2 inhibits HIV-1 replication.
(A) Over expression of TET2 inhibits HIV-1 replication. THP1 cells were transfected with TET2 for 24h and then infected with HIV-1 (Vpr-)/HSA reporter virus at MOI of 0.1. Viral replication was analyzed by HSA reporter expression, n=2.
(B) CRISPR-Cas9 mediated knockout of TET2 in THP1 cells. CTRL cells were transduced by non-targeting gRNA, n=2.
(C) TET2 deletion enhances HIV-1 replication. Control and TET2-KO THP1 cells were infected with HIV-1 (Vpr-, MOI = 0.05). Percentage of infected cells were analyzed at 2 days post infection (dpi) by intracellular p24 FACS (left panel). Different HIV-1 MOIs were used to infect control and TET2-KO THP1 cells and analyzed by intracellular p24 FACS (right panel).
(D) HIV-1 infection and replication kinetics in HIV-1 (Vpr-) infected control and TET2 knockout THP1 cells were measured over 9 days by p24 ELISA. This experiment was repeated twice with same clone and once with another TET2 KO clone, n=2.
(E) Re-expression of TET2 in TET2-KO THP1 cells restored HIV-1 inhibition. Empty vector or vector expressing wildtype or mutant TET2 (1 µg) were transfected into parental or TET2-KO cells and protein expression verified by Western blot 24 hr after transfection. The transfected cells were infected with HIV-1 (Vpr-), and virus replication was analyzed by p24 ELISA at 6 dpi, n=2.
(F) TET2 inhibits HIV-1 replication in human primary macrophages. shRNA-mediated knockdown of TET2 in MDMs and confirmed by RT-qPCR. MDMs were then infected with a macrophagetropic HIV-1 (Vpr-) and viral replication was analyzed by p24 FACS or p24 ELISA (donor n=3). Statistical analysis was performed using Student’s t-test. *, ** and *** indicated a p-value of less than 0.05, 0.01 or 0.001, respectively. n.s= not significant. Error bars were calculated from technical replicates.