21. Kraut, R. & Levine, M. Mutually repressive interactions between the gap genes giant and Kruppel define middle body regions of the Drosophila embryo. Development 111, 611–621 (1991). 22. Struhl, G., Struhl, K. & Macdonald, P. M. The gradient morphogen bicoid is a concentration- dependent transcriptional activator. Cell 57, 1259–1273 (1989). 23. Driever, W. & Nusslein-Volhard, C. The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95–104 (1988). 24. Langeland, J. A., Attai, S. F., Vorwerk, K. & Carroll, S. B. Positioning adjacent pair-rule stripes in the posterior Drosophila embryo. Development 120, 2945–2955 (1994). 25. Hanna-Rose, W. & Hansen, U. Active repression mechanisms of eukaryotic transcription repressors. Trends Genet. 12, 229–234 (1996). 26. Gray, S. & Levine, M. Transcriptional repression in development. Curr. Opin. Cell Biol. 8, 358–364 (1996). 27. Andrioli, L. P., Vasisht, V., Theodosopoulou, E., Oberstein, A. &Small, S. Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms. Development 129, 4931–4940 (2002). 28. Small, S. In vivo analysis of lacZ fusion genes in transgenic Drosophila melanogaster. Methods Enzymol. 326, 146–159 (2000). 29. Struhl, G., Fitzgerald, K. & Greenwald, I. Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74, 331–345 (1993). 30. Lawrence, P. A., Johnston, P., Macdonald, P. &Struhl, G. Borders of parasegmentsin Drosophila embryos are delimited by the fushi tarazu and even-skipped genes. Nature 328, 440–442 (1987). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thank M. Fujioka and J. Jaynes for transgenic flies containing the eve 4 þ 6–lacZ construct; L. Andrioli for discussions and support; A. Oberstein for technical assistance; and C. Desplan, J. Blau and T. Cook for encouragement and comments on the manuscript. D.E.C. was supported by a grant from the NSF. This work was also supported by a grant from the NIH. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to S.S. (stephen.small@nyu.edu). .............................................................. The gene product Murr1 restricts HIV-1 replication in resting CD4 1 lymphocytes Lakshmanan Ganesh 1 , Ezra Burstein 1,2 , Anuradha Guha-Niyogi 1 , Mark K. Louder 1 , John R. Mascola 1 , Leo W. J. Klomp 3 , Cisca Wijmenga 3 , Colin S. Duckett 2 & Gary J. Nabel 1 1 Vaccine Research Center, NIAID, National Institutes of Health, Building 40, Room 4502, MSC-3005, 40 Convent Drive, Bethesda, Maryland 20892-3005, USA 2 University of Michigan, Medical Science I, Room 5315, 1301 Catherine Street, Ann Arbor, Michigan 48109-0602, USA 3 University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands ............................................................................................................................................................................. Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4 1 lymphocytes, the virus repli- cates poorly in resting T cells 1–6 . Factors that block viral replica- tion in these cells might help to prolong the asymptomatic phase of HIV infection 7 ; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation 8,9 , inhibits HIV-1 growth in unstimulated CD4 1 T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kB activity. Knockdown of Murr1 increased NF-kB activity and decreased IkB-a concentrations by facilitating phospho-IkB- a degradation by the proteasome. Murr1 was detected in CD4 1 T cells, and RNA-mediated interference of Murr1 in primary resting CD4 1 lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infec- tion and the progression of AIDS. Murr1 is a highly conserved 190-amino-acid protein that does not have any identifiable motifs, and a homozygous deletion in the gene encoding canine Murr1 leads to copper toxicosis in Bedlington terriers 8 . In this study, Murr1 was initially identified in a two-hybrid screen by binding the X-linked inhibitor of apoptosis, a known activator of NF-kB (refs 10, 11, and E.B., unpublished observations). To study its effect on NF-kB, HIV-1 reporter plasmids with wild- type or mutant (DkB) sites 2 were co-transfected with control or Murr1 expression plasmids in the different cell lines. Murr1 inhib- ited both basal and tumour necrosis factor (TNF)-a-dependent HIV-1 transcription from the wild-type but not the kB-mutant reporter in Jurkat T-leukaemia and 293T renal epithelial cell lines (Fig. 1a, left and middle panels). In contrast, Murr1 did not substantially inhibit tumour growth factor-b-dependent transcrip- tion in HepG2 cells, confirming its specificity (Fig. 1a, right panel). The kB effect was dose-dependent and observed with other inducers of NF-kB, including interleukin-1 (IL-1) and 12-O-tetradecanoyl- phorbol-13-acetate (TPA) (Fig. 1b). Murr1 modulated the expression of endogenous kB-regulated genes: transfection into 293T cells decreased the endogenous cell-surface expression of major histocompatibility complex (MHC) class I, in contrast to CD9, which is independent of NF-kB (Fig. 1c). Its site of action in the NF-kB signalling pathway was further defined by co-transfection of different regulators with an NF-kB reporter in Jurkat T cells. Whereas Murr1 inhibited both IKK-1- and IKK-2-induced NF-kB activity (Fig. 1d, middle and right panels), it failed to block RelA-mediated transcription (Fig. 1d, left panel), indicating that Murr1 might interact downstream of the IkB kinase signalosome. As determined by immunoprecipitation, co- transfected haemagglutinin (HA)-tagged Murr1 and Myc-tagged IKK-2 did not associate in vivo (Fig. 2a, lane 2, left panel). Although IKK-1 also did not associate with Murr1 (data not shown), an interaction between transfected HA-tagged Murr1 and endogenous IkB-a was readily detected (Fig. 2a, lane 6). The ankyrin domain of IkB-a was required for association with Murr1, as were amino acids 1–160 of Murr1 (Supplementary Fig. 1a). A polyclonal antibody against Murr1 demonstrated the associ- ation between endogenous Murr1 and IkB-a in vivo. RelA antibody immunoprecipitated IkB-a,IkB-b and Murr1 (Fig. 2b, lane 10). IkB-a antibody also pulled down RelA and Murr1 (Fig. 2b, lane 12), but the IkB-b antibody did not precipitate Murr1 (Fig. 2b, lane 14), suggesting that Murr1 interacted preferentially with the NF-kB– IkB-a complex. This association was confirmed in vivo by confocal microscopy with fluorescent fusion proteins (Supplementary Fig. 1b), similarly to the pattern of RelA association with IkB-a 12–14 . The physiological consequences of these interactions were deter- mined by knockdown of endogenous Murr1 in 293T cells using control and Murr1-specific short interfering RNA (siRNA) duplexes. The specificity of two such siRNAs, Murr1-1 and Murr1-2, directed to different Murr1 sequences, was first confirmed by transfecting 293T cells with wild-type or mutant siRNAs along with wild-type or mutant Murr1 complementary DNAs modified at the siRNA target site (Supplementary Fig. 2). Transient transfection of Murr1-specific siRNA duplexes downregulated endogenous Murr1 and IkB-a, had little effect on IkB-b, p65 or IKK-2 (Fig. 3a, left panel), and increased kB-dependent reporter activity (Fig. 3a, right panel). To investigate the mechanism of Murr1 action, 293T cells were transfected with a control or Murr1 siRNA. Four days after transfection, cells were treated with the proteasome inhibitor MG132 for 2 h or with vehicle alone and stimulated with TNF-a. Cells depleted of Murr1 showed a decrease in basal IkB-a (Fig. 3a) and an increase and persistence of phospho-IkB-a in response to stimulation with TNF-a (Fig. 3b, left panel). This effect was observed in the absence of a proteasome inhibitor, MG132, but not in its presence (Fig. 3b, right panel), indicating that Murr1 letters to nature NATURE | VOL 426 | 18/25 DECEMBER 2003 | www.nature.com/nature 853 © 2003 Nature Publishing Group