Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.

ABSTRACT

OBJECTIVE: Characterization of the effects of HIV-1 infection and apoptosis on cellular and viral gene expression. METHODS: Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1IIIB-infected CEM-SS T cells. Suppression subtractive hybridization (SSH) was applied to cells from different time points of infection to construct subtracted complementary DNA (cDNA) libraries. Differential screening and Northern blots confirmed differential gene expression and these genes were sequenced and compared with database. RESULTS: T cells undergo apoptosis at early stages of HIV-1IIIB infection (days 5-7 post-infection). Surprisingly, cells begin to recover after day 9 and by day 18 almost all infected cells are viable, even though they maintain the same level of infection. By SSH, differential gene expression profiles between day 7 and day 18 after HIV-1IIIB infection were characterized. SSH yielded two subtracted cDNA libraries; differential screening of the subtracted cDNA libraries suggested that 200 out of 864 colonies were highly expressed at their respective time points. DNA sequence analysis identified specific apoptosis-related genes, HIV-1 viral genes, and other candidate genes of interest. Northern blot analysis confirmed that some of these genes were expressed predominantly at the 'apoptotic' or 'non-apoptotic' time points. CONCLUSIONS: Known and novel cellular gene products have been identified that are directly (or inversely) correlated with apoptosis and may regulate cell death in HIV-1 infection. These results provide a framework for functional studies on the differentially expressed genes and may suggest novel therapeutic approaches for treatment of HIV-1-infected individuals.