No abstract

SUMMARY Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferential targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

Members of the family of DEXH/D-box proteins are involved in all major RNA transactions, including transcription, translation, ribosome biogenesis, and pre-mRNA splicing (1, 2). DEXH/D-box proteins can hydrolyze NTP to NDP in a reaction that is stimulated by, or dependent on, a nucleic acid cofactor. Although several DEXH/D family members exhibit RNA helicase activity in vitro, the action of DEXH/D-box NTPases may not be limited to the unwinding of RNA duplexes. Recent studies suggest that they can act as "RNPases" to displace proteins from nucleic acids (3-6). DEXH-box proteins are defined by conserved motifs I (GXGKT), II (DEXH), III (S/TAT), and VI (QRXGRXGR), which are important for ATP hydrolysis and RNA unwinding (7,8).The DEXH/D-box ATPases Prp5, Brr2, Prp28, Sub2/UAP56, Prp2, Prp16, and Prp22 are involved in pre-mRNA splicing (9). Removal of introns from precursor RNAs is catalyzed by the spliceosome, which is formed by the assembly of U1, U2, and U4/U6/U5 snRNPs and non-snRNP 1 proteins onto the precursor RNA (10, 11). Splicing entails two successive transesterification reactions: in step 1, the 5Ј splice site is cleaved and the branched lariat-intermediate is formed; in step 2, the 3Ј splice site is cleaved and the exons are joined. Mature mRNA is then released, and the spliceosome components are presumed to recycle for the next round of splicing (10). Splice site recognition and positioning of the reactive nucleotides for catalysis requires dynamic remodeling of an intricate network of RNA-RNA and RNA-protein interactions (12, 13). In vitro studies have established that ATP is required for many steps in the splicing cycle and that DEXH/D-box proteins act at those ATPdependent steps (9, 10). For example: Prp28, Brr2, Prp5, and Sub2/UAP56 are important for spliceosome assembly; Prp2 promotes step 1 transesterification; Prp16 is required for the second transesterification step; and Prp22 triggers the release of mature mRNA from the spliceosome (9, 14 -16). Prp2, Prp16, and Prp22 mutants that are defective for ATP hydrolysis are also defective in executing their ATP-dependent functions in pre-mRNA splicing in vitro (16 -19). Such mutations are also invariably lethal in vivo (18,20,21). Moreover, overexpression of non-functional Prp2, Prp16, and Prp22 mutants impairs the growth of wild-type cells (18,20,21). The dominant-negative Prp16 and Prp22 phenotypes can be recapitulated in vitro with purified proteins; for example, inactive Prp16 proteins block step 2 transesterification chemistry and dominant-negative Prp22 proteins block release of mature mRNA from the spliceosome in trans (19). Thus, the steps arrested by the dominant-negative mutants illuminate the function of the wild-type proteins during pre-mRNA splicing. S. cerevisiae PRP43 and its mammalian homologue mDEAH9 were isolated in PCR-based screens for DEAH-box proteins (22,23). Yeast PRP43 is an essential gene that encodes a 767-amino acid polypeptide with a predicted molecular mass of 88 kDa. Arenas and Abelson (22) isolated a temperature-sensit...

SUMMARY A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat as well as via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of, CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.