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Background:  There is an urgent need to explore novel molecular and genetic strategies to slow or halt HIV-1 replication and to prevent viral latency. Cellular proteins that promote infection (e.g., anti-apoptotic proteins) may provide attractive therapeutic targets, particularly if infected cells can be selectively targeted.

Methods:  Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1 IIIB-infected CEM-SS T cells and primary CD4 T cells. Suppression subtractive hybridization was applied to cells from different time points of infection to construct subtracted complementary DNA libraries. Differential screening, Northern blots, and real-time polymerase chain reaction (PCR) confirmed differential gene expression. RNA interference was used to silence DDIT4, using public and in-house programs to design 4 small interfering RNAs (siRNAs) each for green fluorescent protein (GFP) and DDIT4 and their corresponding scrambled siRNA controls. We generated siRNA expression cassettes (SEC) by PCR and transfected the PCR products into T cells using AMAXA^® Nucleofector technology. To increase transfection efficiency, the most effective SEC were cloned into a thymidine analogue cloning vector and titered with their respective controls. Flow cytometry and fluorescence microscopy analyses were performed for GFP siRNAs, and Northern blot analysis was done to assess the DDIT4 silencing effect.

Results:  The gene, DNA-damage-inducible transcript 4 (DDIT4) was cloned from cells surviving long-term HIV-1 infection and is highly conserved. Its homologues, RTP801 (rat) and dig2 (mouse) have been reported to have both pro- and anti-apoptotic activities, depending on the cellular context. Human DDIT4 is a developmentally regulated transcriptional target of p63, p53, and hypoxia-inducible factor-1 (HIF-1). We show that DDIT4 expression is markedly down-regulated and then recovers after HIV-1 IIIB or NL4-3 infection in a human CD4 T-cell line, CEM-SS. DDIT4 expression correlates with cell survival. DDIT4 expression is also down-regulated in IIIB-infected primary CD4 T cells. Using siRNA, we silenced the DDIT4-gene product in primary CD4 T cells and then infected them with HIV-1 IIIB. Our data show that DDIT4 inhibition leads to increased apoptosis of infected T-cells and decreased infection levels in the remaining viable cells.

Conclusions:  Our data suggest that DDIT4 protects HIV-1 infected human CD4 T cells from apoptosis and increases HIV-1 infection.DDIT4 may represent a novel molecular target for drug design.

Keywords: Retroviral pathogenesis; Viral targets and Reservoirs; Host genetic factors