A nucleolar TAR decoy inhibitor of HIV-1 replication

Abstract

The Tat protein is a key regulator of HIV-1 replication (1). Its major function is to transactivate RNA polymerase (pol) II transcription from the viral LTR promoter. The binding of Tat to a transactivation response (TAR) element in the 5Ј UTR of the viral RNAs stimulates the processivity of RNA pol II, greatly increasing HIV-1 RNA transcription (2-6). In the absence of Tat the transcription complex is able to initiate transcription from the LTR, but elongates very inefficiently (3). The transactivation activity of Tat is mediated by the interaction with a positive transcription elongation factor, P-TEFb (7-9), composed of CDK9 and cyclin T1 (10-16). The binding of Tat with cyclin T1 increases its affinity for the TAR element and induces the cooperative binding of the P-TEFb complex to TAR (13). CDK 9 can phosphorylate the carboxyl-terminal domain of the largest subunit of RNA pol II, stimulating its processivity (10,16,17). Because Tat affects one of the earliest stages of HIV-1 gene expression and may be involved in other critical steps in virus replication (reverse transcription for example, ref. 18), it has been considered a good candidate for therapeutic intervention against HIV-1. Among the different gene therapy strategies that have been tested to block Tat activity in human cells one of the most successful is the use of small RNA molecules that work as ''decoys.'' These RNAs mimic the specific RNA binding element for Tat, subsequently leading to its titration. TAR RNA or in vitro-evolved Tat binding aptamers have been previously used as decoys for Tat and have resulted in inhibitory effects on HIV-1 replication (19-23).In addition to its known localization in the nucleoplasm, Tat has been shown to have nucleolar localization properties (24-28). The functional role of Tat nucleolar trafficking is unclear, but it may associate with cyclin T1 in this compartment (29). We have previously used strategies for localizing an anti-HIV ribozyme as well as a Rev binding element in the nucleolus and demonstrated that both of these strategies inhibit HIV-1 replication (30, 31). To test whether Tat nucleolar localization is functionally important, we have used a similar strategy to direct a TAR element into this compartment. We demonstrate here that human T-lymphoblastoid CEM cells stably expressing a nucleolar-localized TAR element are highly resistant to HIV-1 infection. Using in situ hybridization analyses we also show that a Tat-enhanced GFP (EGFP) fusion protein colocalizes with the nucleolar-localized TAR element. The present observations taken together with the nucleolar localization properties of HIV-1 Rev (32-34), as well as some HIV RNAs (30,35,36), represent an additional paradigm for the role of the nucleolus in HIV-1 replication. The potent inhibition of HIV-1 replication mediated by the nucleolar-localized TAR decoy also suggests a strategy for genetic therapy of HIV-1 infection. Materials and MethodsPlasmid Constructs. The U16TAR DNA was prepared synthetically by PCR (37) using the primers A, B, C, D, E...

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

“…Some elegant experiments using a hammerhead ribozyme targeted to the nucleolus and specific for HIV-1 RNA showed that HIV-1 RNA trafficking through the nucleolus is essential for HIV-1 replication (40). Similarly, nucleolar targeting of a RNA-based inhibitor of HIV-1 Tat (a second HIV-1 nucleolar protein) was also able to significantly decrease HIV-1 replication, confirming that nucleolar targeting of Tat is required for HIV-1 replication (41).…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…It has been demonstrated that Tat binds strongly in the nuclei (Fig. 2,3 and 4) and hence its exit from the nucleus would be difficult unless the cell ruptures [135,[142][143][144][145][146][147][148].…”

“…Further, in reality, PTD-based delivery of fusion proteins will invariably result in nuclear targeting [63,135,[142][143][144][145][146][147][148]168] which may not be required in every case. It is useful, however, in some cases such as expression of single chain antibody fragment, where the product is required in the nucleus to inhibit Tat-mediated HIV-LTR transcription [169,170].…”

Relationships between Infectious Titer, Capsid Protein Levels, and Reverse Transcriptase Activities of Diverse Human Immunodeficiency Virus Type 1 Isolates

“…In contrast, the RT assay is an indirect measure of virus titer that involves the use of the endogenous viral RT enzyme to extend an exogenous, ho-mopolymeric DNA primer on an RNA template. In recent literature, approximately 50% of studies comparing the replication efficiencies of different HIV-1 isolates or mutants measured virus titers by TCID 50 assays (10,22,32,33,55), 45% used p24 antigen capture assays (14,21,26,44,49), and Ͻ5% used RT assays (17,27,54). As outlined below, most reports measuring relative in vitro HIV-1 fitness levels by using sensitive competitive replication assays have employed the TCID 50 assay to obtain an accurate measure of virus titers to equalize inocula (3,8,29,34,39,40,43).…”