The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Revinteracting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. HIV-1 utilizes many host cell factors for its replication (1-3), similar to other viruses. There is strong interest in identifying and understanding these components to shed light on the molecular mechanisms of virus replication. Moreover, this can provide the potential for developing new therapeutics. The HIV Rev protein is a key regulator of viral replication that is critical for the late stages of virus replication (4, 5). The bestcharacterized function of Rev involves its potent stimulation of the nuclear export of unspliced and singly spliced ("underspliced") HIV transcripts that encode the viral structural proteins and accessory factors (5). In the absence of Rev, these transcripts are retained in the nucleus because of their incomplete splicing. At the molecular level, Rev binds and oligomerizes along the 351-nt Rev Response Element (RRE) 1 (6) in the env gene that is present in all underspliced HIV transcripts. Rev contains a classical leucine-rich nuclear export sequence that recruits CRM1, a transport receptor of the karyopherin family (7,8). CRM1 commonly is used for nuclear export of cellular proteins, and only infrequently is involved in cellular mRNA export (9, 10). Upon binding to the RRE together with the GTP-bound form of Ran, CRM1 forms the core ...
RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80°C but was almost inactive at 60°C. E stimulated the activity of the core enzyme at 60°C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E , and of archaeal TFE to open complex formation. Opening in the ؊2 and ؊4 region could be achieved by the core enzyme, subunit E stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E -independent role of TFE in the stabilization of open complexes. Transcription in cells of Archaea andBacteria is catalyzed by a single RNA polymerase (RNAP), 2 whereas eukaryotic cells contain three different types of RNAPs (I, II, and III) that carry out specialized functions. Despite their morphological similarity to Bacteria, Archaea have a transcriptional machinery that is more akin to the eukaryotic machinery (1-3).Like eukaryotes, Archaea use extrinsic transcription factors for initiation. The process of transcription initiation in Archaea can be dissected in three steps. The general transcription factor TATA-binding protein (TBP) interacts with the archaeal TATA box, transcription factor B (TFB) stabilizes the binding of TBP to the promoter. The TBP-TFB promoter complex mediates recruitment of RNAP (4, 5). These extrinsic factors show the major structural features of eukaryotic TBP and TFIIB and interact with DNA and RNAP in a similar fashion as their eukaryotic counterparts (6, 7). This exceptional degree of similarity between the archaeal and eukaryotic transcriptional machineries extends also to the RNAP.Archaeal RNAP consist of 11 or 12 different subunits (8 -10) that display a high level of primary sequence similarity to the subunits present in eukaryotic RNAPII. With the exception of subunits RPB8 and RPB9, orthologs of other RNAPII subunits have been identified in all archaeal genomes studied so far. A recent in silico study revealed a high degree of conservation of subunits shared by pol II and Archaea in particular in the regions of RNAP comprising the catalytic center and proteinprotein binding studies showed a similar pattern of subunit interactions between the subunits of pol II and of Pyrococcus RNAP (11). Although very similar to eukaryotic RNAP in subunit composition and transcription initiation factor requirement the archaeal machinery is less complex than the eukaryotic machine...
The HIV-1 Rev protein is essential for the virus because it promotes nuclear export of alternatively-processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in E. coli is a well-behaved folded protein. Binding assays using fluorescently-labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell based assays of HIV-1 production, and provide the first demonstration that recombinant DDX1 binds Rev and RNA, and has RNA dependent catalytic activity.
The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure–function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.
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