A nucleolar TAR decoy inhibitor of HIV-1 replication -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Michienzi et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.212229599.

Materials and Methods
Plasmid Construct.
The TAR DNA was prepared by PCR using an HIV-1 genomic vector (pNL4-3) as substrate for the following primers:

TAR5¢ : 5¢ - GGGGGGTCGACGGTCTCTCTGGTTAGACCA-3¢

TAR3¢ : 5¢ - GGGGTCTAGAGGGCACACACTACTTTGAGC-3¢ .

The PCR product was digested with SalI and XbaI restriction enzymes and subcloned into the corresponding sites of the pTz/U6+1 expression cassette (1-3), generating the U6+1/TAR construct.

p24^GagAnalysis.
p24^Gag analyses were performed as observed (4). Results
Intracellular Expression and Localization of the TAR Decoy.
To further investigate the contribution of the nucleolar localization of the U16TAR decoy toward its anti-HIV-1 activity, we compared the anti-HIV-1 activites of the U16TAR decoy with a TAR RNA decoy that localizes only to the nucleus. The nuclear TAR decoy includes the first 111 bp of the HIV-1 transcript and was expressed from the U6 promoter (U6+1/TAR plasmid, refs. 1-3). The expression cassette for this construct also contains a stem-loop sequence followed by five thymidines (for pol III termination) positioned downstream of the TAR element. The U6+1/TAR plasmid was transiently transfected into human 293 cells and total RNA was isolated 48 h posttransfection. Northern blot analyses revealed strong expression of the TAR decoy RNA during transient transfection (data not shown). In situ hybridization analyses were carried out on the U6+1/TAR-transfected cells to visualize the intracellular localization of the decoy. The CY3-labeled probe for the decoy results in red fluorescence (Fig. 5, TAR panel), whereas endogenous U3 snoRNA (for nucleolar visualization) was probed with an Oregon green 488-labeled probe (Fig. 5, U3 panel). The TAR signal is diffusely distributed throughout the nucleus with bright granules or foci of accumulation (Fig. 5, TAR panel). There is virtually no overlap between the TAR and U3 fluorescent signals (Fig. 5, TAR/U3 panel), indicating that the TAR element is localized primarily within the nucleoplasm.

To evaluate the relative antiviral activities of the U16TAR and U6+1/TAR decoys, 293 cells were transiently cotransfected with either U6+1/TAR, U6+1/U16TAR, or the U6+1 backbone plasmid and the HIV-1 genomic clone pNL4-3. As a measure of viral replication, HIV-1 p24^Gag was measured at 1, 2, 3, and 4 days posttransfection (Fig. 6). Peak viral production is observed at day 3 in transfection assays. The strongest inhibition of HIV-1 p24^Gag was observed in cells cotransfected with the U6+1/U16TAR construct, with no detectable p24^Gag. Moderate inhibition was observed with the U6+1/TAR decoy, which is consistent with the ability of such decoys to functionally compete for Tat (5-9). The data presented in Fig. 6 represent the average values from duplicate transfections, and the standard errors for each point are around 5% maximum.

Discussion

We have demonstrated that a nucleolar-localized TAR element is a potent inhibitor of HIV-1 replication. Comparison of the nucleolar versus nuclear-localized decoys revealed that nucleolar localization results in stronger inhibitory activity than nucleoplasmic localization. These results may reflect more efficient titration of Tat in the nucleolus because of its early trafficking into this organelle (10), or alternatively it may indicate that nucleolar Tat may be in a form that is critical for efficient transcriptional activation of HIV-1 transcription (11). Sequestering of Tat in the nucleolus presumably retards its ability to function in transcriptional activation in the nucleoplasm. Nevertheless, it is difficult to draw definitive conclusions from this type of comparative analysis because several variables such as the stability and overall structure of the two TAR elements may also be involved in the observed differential inhibitory activities.

References

1. Good, P. D., Krikos, A. J., Li, S. X., Bertrand, E., Lee, N. S., Giver, L., Ellington, A., Zaia, J. A. Rossi, J. J. & Engelke, D. R. (1997) Gene Ther. 4, 45–54.

2. Kunkel, G. R. & Pederson, T. (1988) Genes Dev. 2, 196–204.

3. Kunkel, G. R. & Pederson, T. (1989) Nucleic Acids Res. 17, 7371–7379.

4. Michienzi, A., Cagnon, L., Bahner, I. & Rossi, J. J. (2000) Proc. Natl. Acad. Sci. USA 97, 8955–8960.

5. Sullenger, B. A., Gallardo, H. F., Ungers, G. E. & Gilboa, E. (1990) Cell 63, 601–608.

6. Sullenger, B. A., Gallardo, H. F., Ungers, G. E. & Gilboa, E. (1991) J. Virol. 65, 6811–6816.

7. Lisziewicz, J., Sun, D., Smythe, J., Lusso, P., Lori, F., Louie, A., Markham, P., Reitz, M. & Gallo, R. C. (1993) Proc. Natl. Acad. Sci. USA 90, 8000–8004.

8. Lee, S. W., Gallardo, H. F., Gaspar, O., Smith, C. & Gilboa, E. (1995) Gene Ther. 2, 377–384.

9. Browning, C. M., Cagnon, L., Good, P. D, Rossi, J., Engelke, D. R. & Markovitz, D. M. (1999) J. Virol. 73, 5191–5195.

10. Luznik, L., Martone, M. E., Kraus, G., Zhang, Y., Xu, Y., Ellisman, M. H. & Wong-Staal, F. (1995) AIDS Res. Hum. Retroviruses 11, 795–804.

11. Marcello, A., Cinelli, R. A., Ferrari, A., Signorelli, A., Tyagi, M., Pellegrini, V., Beltram, F. & Giacca, M. (2001) J. Biol. Chem. 276, 39220–39225.