64   Actinomycin D Enhances HIV-1 Replication via an Interleukin-9 Dependent Mechanism.

T. Imamichi*¹, S. C. Berg¹, R. A. Lempick¹, L. D. Palmer¹, M. W. Baseler¹, J. W. Adelsberger¹, C. M. Watkins¹, M. A. Murphy¹, E. L. Nelson¹, D. A. Prieto¹, J. Guo², J. G. Levin², and H. C. Lane³.
¹SAIC-Frederick/NCI-FCRDC, Frederick;²NICHD/NIH, Bethesda; and³NIAID/NIH, Bethesda, MD.

Background:Actinomycin D (ActD), a transcription inhibitor, has been reported as a potential suppressor of HIV-1 replication. In this study, we evaluated the impact of ActD on HIV replication in tissue culture.

Methods:MT-2, MT-4, and Jurkat cells and PBMC were infected with a wild-type HIV-1, NL4.3, and then cultured for seven days in the presence of varying concentrations of ActD. Replication capacity was measured by the p24 antigen capture assay.

Results:High concentrations of ActD (>100 nM) demonstrated a strong cytotoxicity in all cells tested. Concentrations of 1 to 10 nM Act D upregulated HIV replication (8-fold) in MT-2 cells, despite down-regulation of CD4 and CXCR4 expression on the cell surface. Pretreatment of MT-2 cells with 200 nM ActD before infection enhanced HIV replication in MT-2 cells by 6-fold. Interestingly, the conditioned medium (CM) derived from the pretreated MT-2 cells also enhanced HIV replication. These results suggested that the ActD treatment led to production of a soluble factor(s) that subsequently enhanced replication. To attempt to determine what factor(s) might be responsible for this upregulation of HIV replication, DNA microarrays were used to analyze patterns of gene expression in MT-2 cells treated with Act D compared to untreated cells. Cells treated with Act D showed a 74-fold increase in interleukin-9 (IL-9) gene transcription, and the expression was confirmed by RNase protection assay. Immunoabsorption of IL-9 from the CM suppressed the augmentation of HIV replication by the CM, even though recombinant IL-9 alone did not lead to significant effects on HIV replication.

Conclusions:These results suggest that IL-9, in combination with an as yet unidentified factor(s) in the CM derived from Act D treated MT-2 cells, is capable of enhancing HIV replication. The data further imply that expression of the IL-9 gene is under the influence of a potent inhibitor(s) of transcription.