The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

HTLV-1 is an oncogenic human retrovirus and the etiologic agent of the highly aggressive ATL malignancy. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in the pathogenic process. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral lifecycle and directly contributes to its pathogenic potential. Herein, we identified the cellular transcription factor YBX1 as a binding partner for HBZ. We found YBX1 activated transcription and enhanced Tax-mediated transcription from the viral 5' LTR promoter. Interestingly, YBX1 also interacted with Tax. shRNA-mediated loss of YBX1 decreased transcript and protein abundance of both Tax and HBZ in HTLV-1-transformed T-cell lines, as well as Tax association with the 5' LTR. Conversely, YBX1 transcriptional activation of the 5' LTR promoter was increased in the absence of HBZ. YBX1 was found to be associated with both the 5' and 3' LTRs in HTLV-1-transformed and ATL-derived T-cell lines. Together, these data suggest that YBX1 positively influences transcription from both the 5' and 3' promoter elements. YBX1 is able to interact with Tax and help recruit Tax to the 5' LTR. However, through interactions with HBZ, YBX1 transcriptional activation of the 5' LTR is repressed.

Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection.

Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.

Sec24C is an HIV-1 host dependency factor crucial for virus replication.

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus-host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and identified Sec24C as an HIV-1 host dependency factor. Gene deletion and complementation in Jurkat cells revealed that Sec24C facilitates infection and markedly enhances HIV-1 spreading infection. Downregulation of Sec24C in HeLa cells substantially reduced HIV-1 core stability and adversely affected reverse transcription, nuclear import and infectivity. Live-cell microscopy showed that Sec24C co-trafficked with HIV-1 cores in the cytoplasm during virus ingress. Biochemical assays demonstrated that Sec24C directly and specifically interacted with hexameric capsid lattices. A 2.3-Å resolution crystal structure of Sec24C228-242 in the complex with a capsid hexamer revealed that the Sec24C FG-motif bound to a pocket comprised of two adjoining capsid subunits. Combined with previous data1-4, our findings indicate that a capsid-binding FG-motif is conserved in unrelated proteins present in the cytoplasm (Sec24C), the nuclear pore (Nup153; refs. 3,4) and the nucleus (CPSF6; refs. 1,2). We propose that these virus-host interactions during HIV-1 trafficking across different cellular compartments are crucial for productive infection of target cells.

Structural and mechanistic bases for a potent HIV-1 capsid inhibitor.

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

HTLV-1 Tax-1 interacts with SNX27 to regulate cellular localization of the HTLV-1 receptor molecule, GLUT1.

An estimated 10-20 million people worldwide are infected with human T cell leukemia virus type 1 (HTLV-1), with endemic areas of infection in Japan, Australia, the Caribbean, and Africa. HTLV-1 is the causative agent of adult T cell leukemia (ATL) and HTLV-1 associated myopathy/tropic spastic paraparesis (HAM/TSP). HTLV-1 expresses several regulatory and accessory genes that function at different stages of the virus life cycle. The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products, and has also been demonstrated to play a key role in the pathogenesis of the virus. Several studies have identified a PDZ binding motif (PBM) at the carboxyl terminus of Tax-1 and demonstrated the importance of this domain for HTLV-1 induced cellular transformation. Using a mass spectrometry-based proteomics approach we identified sorting nexin 27 (SNX27) as a novel interacting partner of Tax-1. Further, we demonstrated that their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domains. SNX27 has been shown to promote the plasma membrane localization of glucose transport 1 (GLUT1), one of the receptor molecules of the HTLV-1 virus, and the receptor molecule required for HTLV-1 fusion and entry. We postulated that Tax-1 alters GLUT1 localization via its interaction with SNX27. We demonstrate that over expression of Tax-1 in cells causes a reduction of GLUT1 on the plasma membrane. Furthermore, we show that knockdown of SNX27 results in increased virion release and decreased HTLV-1 infectivity. Collectively, we demonstrate the first known mechanism by which HTLV-1 regulates a receptor molecule post-infection.

Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5.

Human T-cell leukemia virus type 1 (HTLV-1) encodes a protein derived from the antisense strand of the proviral genome designated HBZ (HTLV-1 basic leucine zipper factor). HBZ is the only viral gene consistently expressed in infected patients and adult T-cell leukemia/lymphoma (ATL) tumor cell lines. It functions to antagonize many activities of the Tax viral transcriptional activator, suppresses apoptosis, and supports proliferation of ATL cells. Factors that regulate the stability of HBZ are thus important to the pathophysiology of ATL development. Using affinity-tagged protein and shotgun proteomics, we identified UBR5 as a novel HBZ-binding partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Herein, we investigated the role of UBR5 in HTLV-1-mediated T-cell transformation and leukemia/lymphoma development. The UBR5/HBZ interaction was verified in vivo using over-expression constructs, as well as endogenously in T-cells. shRNA-mediated knockdown of UBR5 enhanced HBZ steady-state levels by stabilizing the HBZ protein. Interestingly, the related HTLV-2 antisense-derived protein, APH-2, also interacted with UBR5 in vivo. However, knockdown of UBR5 did not affect APH-2 protein stability. Co-immunoprecipitation assays identified ubiquitination of HBZ and knockdown of UBR5 resulted in a decrease in HBZ ubiquitination. MS/MS analysis identified seven ubiquitinated lysines in HBZ. Interestingly, UBR5 expression was upregulated in established T lymphocytic leukemia/lymphoma cell lines and the later stage of T-cell transformation in vitro. Finally, we demonstrated loss of UBR5 decreased cellular proliferation in transformed T-cell lines. Overall, our study provides evidence for UBR5 as a host cell E3 ubiquitin-protein ligase responsible for regulating HBZ protein stability. Additionally, our data suggests UBR5 plays an important role in maintaining the proliferative phenotype of transformed T-cell lines.

Dynamic Interconversions of HCV Helicase Binding Modes on the Nucleic Acid Substrate.

The dynamics involved in the interaction between hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase and its nucleic acid substrate have been the subject of interest for some time given the key role of this enzyme in viral replication. Here, we employed fluorescence-based techniques and focused on events that precede the unwinding process. Both ensemble Förster resonance energy transfer (FRET) and ensemble protein induced fluorescence enhancement (PIFE) assays show binding on the 3' single-stranded overhang of model DNA substrates (>5 nucleotides) with no preference for the single-stranded/double-stranded (ss/ds) junction. Single-molecule PIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases. The enzyme is able to transition between binding sites in both directions without dissociating from the nucleic acid. In contrast, the NS3 mutant W501A, which is unable to engage in stacking interactions with the DNA, is severely compromised in this switching activity. Altogether our data are consistent with a model for NS3 dynamics that favors ATP-independent random binding and sliding by one and two nucleotides along the overhang of the loading strand.

Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.

Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.

Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap.

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5ADII) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5ADII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5ADII. These experiments have led to the identification of an RNA binding motif (305RSRKFPR311) in an arginine-rich region of NS5ADII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5ADII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5ADII modulates the activity of NS5B and potentially regulates HCV replication.

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.

While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.

Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5'-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains - pentatricopeptide repeat (PPR) and metallonuclease (NYN) - that are present in some other RNA processing factors. Here, we combined chemical modification of lysines and multiple-reaction monitoring mass spectrometry to identify putative substrate-contacting residues in Arabidopsis thaliana PRORP1 (AtPRORP1), and subsequently validated these candidate sites by site-directed mutagenesis. Using biochemical studies to characterize the wild-type (WT) and mutant derivatives, we found that AtPRORP1 exploits specific lysines strategically positioned at the tips of it's V-shaped arms, in the first PPR motif and in the NYN domain proximal to the catalytic center, to bind and cleave pre-tRNA. Our results confirm that the protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unanticipated parallel in their mode of action.

Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

Identification and characterization of HTLV-1 HBZ post-translational modifications.

Human T-cell leukemia virus type-1 (HTLV-1) is estimated to infect 15-25 million people worldwide, with several areas including southern Japan and the Caribbean basin being endemic. The virus is the etiological agent of debilitating and fatal diseases, for which there is currently no long-term cure. In the majority of cases of leukemia caused by HTLV-1, only a single viral gene, hbz, and its cognate protein, HBZ, are expressed and their importance is increasingly being recognized in the development of HTLV-1-associated disease. We hypothesized that HBZ, like other HTLV-1 proteins, has properties and functions regulated by post-translational modifications (PTMs) that affect specific signaling pathways important for disease development. To date, PTM of HBZ has not been described. We used an affinity-tagged protein and mass spectrometry method to identify seven modifications of HBZ for the first time. We examined how these PTMs affected the ability of HBZ to modulate several pathways, as measured using luciferase reporter assays. Herein, we report that none of the identified PTMs affected HBZ stability or its regulation of tested pathways.

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase.

BACKGROUND: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. RESULTS: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. CONCLUSIONS: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.

A critical role of the C-terminal segment for allosteric inhibitor-induced aberrant multimerization of HIV-1 integrase.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.

Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.

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.