HIV-1 Integrase-DNA Recognition Mechanisms

Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review

An Allosteric Mechanism for Inhibiting HIV-1 Integrase with a Small Molecule

HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN

Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c

Mouse Dux is myotoxic and shares partial functional homology with its human paralog DUX4

D4Z4 repeats are present in at least 11 different mammalian species, including humans and mice. Each repeat contains an open reading frame encoding a double homeodomain (DUX) family transcription factor. Aberrant expression of the D4Z4 ORF called DUX4 is associated with the pathogenesis of Facioscapulohumeral muscular dystrophy (FSHD). DUX4 is toxic to numerous cell types of different species, and over-expression caused dysmorphism and developmental arrest in frogs and zebrafish, embryonic lethality in transgenic mice, and lesions in mouse muscle. Because DUX4 is a primate-specific gene, questions have been raised about the biological relevance of over-expressing it in non-primate models, as DUX4 toxicity could be related to non-specific cellular stress induced by over-expressing a DUX family transcription factor in organisms that did not co-evolve its regulated transcriptional networks. We assessed toxic phenotypes of DUX family genes, including DUX4, DUX1, DUX5, DUXA, DUX4-s, Dux-bl and mouse Dux. We found that DUX proteins were not universally toxic, and only the mouse Dux gene caused similar toxic phenotypes as human DUX4. Using RNA-seq, we found that 80% of genes upregulated by Dux were similarly increased in DUX4-expressing cells. Moreover, 43% of Dux-responsive genes contained ChIP-seq binding sites for both Dux and DUX4, and both proteins had similar consensus binding site sequences. These results suggested DUX4 and Dux may regulate some common pathways, and despite diverging from a common progenitor under different selective pressures for millions of years, the two genes maintain partial functional homology

Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

<div><p>The <i>DUX4</i> gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the <i>DUX4</i> open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the <i>DUX4</i> cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of <i>DUX4</i>-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged <i>DUX4</i> ORF, and the natural 3’ untranslated region (pLAM) harboring two small introns, <i>DUX4</i> exons 2 and 3, and the non-canonical poly A signal required for stabilizing <i>DUX4</i> mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and <i>DUX4</i> stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.</p></div

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy

AAV.D4Z4.V5 vectors are non-toxic compared to AAV.CMV.DUX4.V5.

<p>H&E stained cryosections of tibialis anterior muscles isolated from C57BL/6 mice, 2 weeks after injection with 3x10¹⁰ particles of the indicated AAV6 vectors, or saline. Top panels (A) show low power images, and (B) are high power images of the same sections. Arrows point out examples of myofibers with centrally located nuclei, which are a histological indication that muscles were damaged and subsequently repaired. Note that AAV.DUX4.V5 vectors cause widespread muscle damage and regeneration, while AAV.D4Z4.V5 and saline did not.</p

Schematic of chromosome 4, D4Z4, and DUX4-expressing AAV vectors.

<p>A: A representation of the telomeric region of the chromosome 4 long arm (4q35). Drawing is not to scale. The 4q35 subtelomere harbors polymorphic, 3.3 kb D4Z4 repeat arrays, as well as other genes, some of which are indicated. This region is normally embedded in repressive heterochromatin. Contraction of the D4Z4 repeat array (in FSHD1) or mutations in SMCHD1 (in FSHD2) leads to epigenetic changes in the 4q35 region, and subsequently permits transcription of the DUX4 gene. An “FSHD permissive” haplotype creates a polyA signal in the pLAM region located downstream of the array. DUX4 transcripts initiated in the last D4Z4 unit extend to this signal and are stabilized by a polyA tail, thereby allowing the mRNA to be translated into the toxic, pro-apoptotic DUX4 protein. B: Two different AAV vectors were engineered to express DUX4. The first generation vector utilized a CMV promoter and SV40 polyA signal. The DUX4 ORF was tagged at the 3’ end with sequences encoding a V5 tag, thereby producing a full-length DUX4 protein containing a carboxy-terminal V5 epitope fusion. ITR, AAV2 inverted terminal repeats. The second generation AAV.D4Z4.V5 vector essentially recapitulates the terminal D4Z4 repeat and pLAM sequences isolated from an FSHD patient, but engineered to express DUX4 with a carboxy-terminal V5 epitope fusion.</p

The DUX4-55aa protein expressed from mis-spliced DUX4 V5 mRNA does not activate apoptosis in vitro.

<p>(A) Western blot confirms expression of DUX4.V5, DUX4-55aa, and DUX4.HOX1.V5 constructs in HEK293 cells transfected two days earlier. The DUX4-55aa construct lacked a V5 tag, and proteins were therefore detected using rabbit anti-DUX4 primary antibodies followed by HRP-coupled goat-anti-rabbit secondary antibodies. DUX4-55aa is ~6 kDa larger than full-length DUX4, which migrates at ~52 kDa. (B) DUX4-55aa protein does not activate apoptosis in vitro. HEK293 cells were transfected with 200 ng of the indicated CMV expression plasmids and plated simultaneously on 96-well plates. Caspase-3/7 activity was measured 48 hours later using a fluorescent plate reader. High caspase-3/7 activity in CMV.DUX4.V5-transfected HEK293 cells indicated that the DUX4.V5 protein caused apoptotic cell death, as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref008" target="_blank">8</a>]. The non-toxic DUX4.HOX1.V5 protein, which was engineered to lack DNA binding function, failed to induce apoptosis, as expected [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref008" target="_blank">8</a>]. Likewise, CMV.DUX4-55aa did not activate caspase-3/7. *, indicates significant difference from DUX4.V5-treated cells. Data represent means +/- sem, averaged from 6 separate samples performed from two independent experiments. UNT, untransfected.</p