It has been demonstrated previously that the transcriptional repressor domain called the Krüppel-associated box (KRAB), conserved in a large number of Krüppel-type zinc finger proteins, fused to Tat transdominant negative mutants, is able to silence HIV-1 long terminal repeat (LTR)-driven gene expression in transient transfection assays. In the present study chimeric Tat mutant-KRAB retroviral expression vectors were used to control HIV-1 replication in acutely infected cells. It was found that while transient and stable expression of Tat mutant-KRAB chimeric proteins represses HIV-1 LTR-driven gene transcription in transient assays, stable expression of Tat mutant-KRAB chimeric molecules does not confer resistance to HIV-1 infection in Jurkat T lymphocytic cell lines. The results provide further evidence that transient transfection may underestimate the role of chromosomal structure in transcriptional regulation and highlight the caveat of direct extrapolation of transient results for designing gene therapy strategies for efficient control of HIV-1 infection.