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Ribozyme-Mediated Inhibition of HIV 1 Suggests Nucleolar Trafficking of HIV-1 RNA

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Ribozyme-Mediated Inhibition of HIV 1 Suggests Nucleolar Trafficking of HIV-1 RNA Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA Alessandro Michienzi*, Laurence Cagnon*, Ingrid Bahner*, and John J. Rossi*†‡ *Department of Molecular Biology, Beckman Research Institute of the City of Hope, and †Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010-3011 Communicated by Arthur Landy, Brown University, Providence, RI, May 30, 2000 (received for review April 22, 2000) The HIV regulatory proteins Tat and Rev have a nucleolar localiza- Ribozymes are RNAs with catalytic activity (28). The ham- tion property in human cells. However, no functional role has been merhead ribozyme is the simplest in terms of size and structure attributed to this localization. Recently it has been demonstrated and can readily be engineered to perform intermolecular cleav- that expression of Rev induces nucleolar relocalization of some age on targeted RNA molecules. These properties make this protein factors involved in Rev export. Because the function of Rev ribozyme a useful tool for inactivating gene expression and a is to bind HIV RNA and facilitate transport of singly spliced and potential therapeutic agent. Moreover, ribozymes can be very unspliced RNA to the cytoplasm, it is likely that the nucleolus plays effective inhibitors of gene expression when they are colocalized a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 with their target RNAs (29, 30). We have taken advantage of RNAs into the nucleolus, a hammerhead ribozyme that specifically ribozyme-mediated inactivation of targeted RNAs to investigate cleaves HIV-1 RNA was inserted into the body of the U16 small whether there is nucleolar trafficking of HIV RNA. To deter- nucleolar RNA, resulting in accumulation of the ribozyme within mine whether this trafficking takes place, a hammerhead ri- ؉ the nucleoli of human cells. HeLa CD4 and T cells expressing this bozyme targeted to a highly conserved sequence in the U5 region nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 of HIV-1 was delivered into the nucleoli of human cells. The replication. The results presented here suggest a trafficking of ability of this nucleolar-localized ribozyme to inhibit the repli- HIV-1 RNA through the nucleoli of human cells, thus posing a cation of HIV-1 was demonstrated in HeLa CD4ϩ and CEM T different paradigm for lentiviral RNA processing. lymphocytes, which are markedly resistant to HIV-1 infection as a consequence of ribozyme-mediated cleavage of HIV-1. These he expression of HIV type 1 (HIV-1) is controlled by a results can be best explained by nucleolar trafficking of HIV Tposttranscriptional mechanism. From a single primary tran- RNA, making them susceptible targets for ribozyme-mediated script several mRNAs are generated. These RNAs can be divided inhibition. The inhibition of HIV by a nucleolar localized into three main classes: unspliced 9-kb, singly spliced 4-kb, and the ribozyme has important implications for the biology and patho- multiply spliced 2-kb RNAs. Each of these RNAs is exported to the genesis of HIV-1 infection, as well as therapeutic potential. cytoplasm for translation and, in the case of the 9-kb RNA, for packaging into virions (1). Normally, pre-mRNAs must undergo a Materials and Methods splicing process to remove one or more introns before being Plasmid Constructs. The U16Rz was prepared synthetically by exported to the cytoplasm. HIV-1 overcomes this limitation, al- PCR (31) using the primers A, B, C, D, E, and F: lowing singly spliced and unspliced RNA to be exported via A: 5Ј-CTTGCAATGATGTCGTAATTTGCGTCTTAC- interaction with its own encoded Rev protein. This regulatory TCTGTTCTCAGCGACAGTTGAA-3Ј protein binds an RNA stem-loop structure termed the Rev re- B: 5Ј-TGTGCCCGTTTCGTCCTCACGGACTCATCA(C͞ sponse element located within the env coding region of singly G)TGTTGTGTGATTTTCAACTGTCGCTGAG-3Ј spliced and unspliced HIV RNAs (2–5). Binding of Rev to this C: 5Ј-GGACGAAACGGGCACACAAAACCTGCTGT- element promotes the export, stability, and translation of these CAGTAAGCTGGTACAGAAGGTTG-3Ј HIV-1 RNAs (6–15). The export process is mediated by the nuclear D: 5Ј-TTTCTTGCTCAGTAAGAATTTTCGTCAAC- export signal of Rev, which binds the receptor exportin 1͞CRM1. CTTCTGTACCAGCTTACTGAC-3Ј It is believed that CRM1 bridges the interaction of Rev with the E: 5Ј-CCCCCGAGCTCCTTGCAATGATGTCGTAA-3Ј nucleoporins required for export to the cytoplasm (16). F: 5Ј-CCCCCCAAGCTTTTTCTTGCTCAGTAAGAA-3Ј When Rev and Tat are expressed independently of other HIV The B primer contains an equimolar mixture of C and G transcripts, these proteins localize within the nucleolus of human nucleotides at the position indicated to simultaneously generate cells (17–22). The simultaneous presence of a nuclear export signal by PCR the wild-type (wt) and mutant versions of U16Rz. The ͞ as well as a nuclear import localization signal confers upon Rev the PCR product was subcloned in the SacI and HindIII sites of the ability to shuttle between the nucleus and the cytoplasm (16). It has pGEM 9zf(Ϫ) vector (Promega), giving rise to the pGEM͞ recently been reported that in HeLa cells, the expression of Rev U16Rz wt and mutant constructs. These constructs were linear- induces the relocalization of the nucleoporins Nup98 and Nup214, ized with HindIII and transcribed in vitro to test their catalytic along with a significant fraction of CRM1, into the nucleolus (23). activity against an RNA oligonucleotide containing the HIV-1 This result has led to the hypothesis that formation of the Rev- RNA target (data not shown). CRM1-nucleoporin complex targeted to the nuclear pore complex The U16Rz wt and mutant sequences were amplified by PCR BIOCHEMISTRY occurs in the nucleolus. It can be similarly hypothesized that HIV RNAs are also relocalized to the nucleolus before cytoplasmic export. Previous studies, which used in situ hybridization assays to Abbreviations: wt, wild type; LTR, long terminal repeat; snoRNA, small nucleolar RNA; moi, define the subcellular localization of HIV RNAs, failed to detect multiplicity of infection. these RNAs in the nucleoli (24–27). This failure to detect these ‡To whom correspondence should be addressed at: Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010-3011. E-mail: jrossi@ RNAs is most likely due to the dynamic process of RNA transport, coh.org. making it difficult to identify discrete nucleolar localization. There- The publication costs of this article were defrayed in part by page charge payment. This fore we have investigated the same problem, using an alternative article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. strategy based on the use of nucleolar localized ribozymes. §1734 solely to indicate this fact. PNAS ͉ August 1, 2000 ͉ vol. 97 ͉ no. 16 ͉ 8955–8960 Downloaded by guest on September 28, 2021 from the pGEM͞U16Rz wt and mutant constructs, using the infection. p24 values were determined with the HIV-1 p24 SalI5Ј and XbaI3Ј primers: antigen capture assay (Science Applications International Corp., SalI5Ј:5Ј- CCCCCCCCGTCGACCTTGCAATGAT- Frederick, MD) according to the manufacturer’s instructions. GTCGTAATTTG-3Ј CEM cells (2.5 ϫ 105) derived from stable clones were infected Ј Ј XbaI3:5- CCCCTCTAGAAAAAATTTCTTGCTCAG- with HIV-1NL4–3 at a multiplicity of infection (moi) of 0.0002 TAAGAATTT-3Ј (low moi experiment, Fig. 4B) and 0.002 (high moi experiment, The PCR products were ligated in the SalI and XbaI sites of Fig. 5). Infections were performed in duplicate. After infection, the pTz͞U6ϩ1 expression cassette (32) generating the U6ϩ1͞ the cells were resuspended in 12 ml of complete medium, and U16Rz wt and mutant constructs. The pTz͞U6ϩ1 cassette p24 accumulation in the supernatant was monitored over time. contains the U6 promoter that allows transcription driven by The cells were split and refed twice a week. The p24 analyses RNA pol III but does not contain the sequence elements were performed using the HIV-1 p24 antigen capture assay kit necessary for 5Ј capping, which might interfere with the nucle- (Science Applications International Corp. Frederick). For the olar localization encoded in the C͞D motif of U16. Six thymi- high moi experiment the p24 analyses were performed on dines were added at the 3Ј end of the ribozyme coding sequence days 7 and 11 (data not shown), after which the pathogenic to terminate RNA polymerase III transcription. effects of the virus on the control cells made p24 determinations The BamHI–XbaI fragments from U6ϩ1͞U16Rz wt and irrelevant. mutant constructs (containing the U6 promoter) were filled in, and the resulting fragment was inserted in both orientations into RNA Preparation and Northern Blot Analyses. Total RNA was pre- the NheI site of the pBabe puro retroviral vector [U3 region of pared using the RNA-STAT 60 reagent (Tel-Test ‘‘B’’; Tel-Test, the 3Ј long terminal repeat (LTR)], giving rise to the following Friendswood, TX) according to the manufacturer’s protocol. The constructs: pBabe puro͞U16Rz F (wt and mutant) and pBabe RNA was electrophoresed in a 1% agarose͞formaldehyde gel or a puro͞U16Rz R (wt and mutant) (see Fig. 3A). 6% polyacrylamide͞7 M urea gel and blotted onto a nylon filter. To simultaneously detect the U16Rz RNA and the endogenous U16 Cell Culture. 293 (American Type Culture Collection: CRL 1573) small nucleolar RNA (snoRNA), we used a radiolabeled probe and HeLa-CD4-LTR-␤-gal cells (obtained from the AIDS Re- complementary to the 3Ј end of U16 (Fig. 4A Left). To detect only search and Reference Reagent Program no. 1470) were both the U16Rz RNA we used a probe complementary to the hammer- propagated in Dulbecco’s modified Eagle’s medium with high head ribozyme sequence. To detect the loading controls, we used ͞ ␤ Lys glucose (Irvine Scientific) supplemented with 10% FBS (GIBCO probes specific for -actin mRNA and tRNA3 . BRL), 200 mM L-glutamine (Irvine Scientific), 10 units͞ml peni- cillin G (GIBCO͞BRL), 10 mg͞ml streptomycin (GIBCO͞BRL), In Situ Hybridization.
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