Host cell interactome of HIV-1 Rev includes RNA helicases involved in multiple facets of virus production.

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 Rev-interacting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. The strategy used here for identifying Rev interaction partners should prove effective for analyzing other viral and cellular proteins.

Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement.

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.

Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases.

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure-function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the alpha subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the DeltaH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication.

The human immunodeficiency virus type 1 (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 Escherichia 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.

Structure of an archaeal RNA polymerase.

Related multisubunit RNA polymerases (RNAPs) carry out gene transcription in all kingdoms of life. Since structural information is limited to bacterial and eukaryotic RNAPs, we determined the cryo-electron microscopic structure of the RNAP from the thermophilic archaeon Pyrococcus furiosus at 13 A resolution. Comparison with eukaryotic RNAP II reveals a conserved architecture, no homologues for subunits Rpb8 and Rpb9, and significant deviation in the polymerase foot, jaws, pore, and protrusion. The structural organization of the archaeal RNA polymerase serves as a reference for future structure-function analysis of the transcription mechanism and allows for evolutionary comparisons.

The RPB7 orthologue E' is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation.

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 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the core enzyme at 60 degrees 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 factor E is a part of transcription elongation complexes.

A homologue of the N-terminal domain of the alpha subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions -9 and -11. In complexes stalled at +20, TFE cross-linked specifically to positions +9, +11, and +16 of the non-template strand. Analyses of transcription complexes stalled at position +20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.

Protein-protein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors.

Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.

The role of transcription initiation factor IIIB subunits in promoter opening probed by photochemical cross-linking.

The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening. TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter. Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as sigma subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes. Brf1, Bdp1, and several pol III subunits can be cross-linked to the nontranscribed strand of the U6 promoter at base pair -9/-8 and +2/+3 (relative to the transcriptional start as +1), respectively the upstream and downstream ends of the DNA segment that opens up into the transcription bubble. Cross-linking of Bdp1 and Brf1 is detected at 0 degrees C in closed preinitiation complexes and at 30 degrees C in complexes that are partly open, but also it is detected in mutant TFIIIB-pol III-DNA complexes that are unable to open the promoter. In contrast, promoter opening-defective TFIIIB mutants generate significant changes of cross-linking of polymerase subunits. The weight of this evidence argues in favor of an indirect mode of action of TFIIIB in promoter opening.