Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.

Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome.

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

Detection of Pb(II): Au Nanoparticle Incorporated CuBTC MOFs.

In the present investigation, copper benzene tricarboxylate metal organic frameworks (CuBTC MOF) and Au nanoparticle incorporated CuBTC MOF (Au@CuBTC) were synthesized by the conventional solvothermal method in a round bottom flask at 105°C and kept in an oil bath. The synthesized CuBTC MOF and Au@CuBTC MOFs were characterized by structure using X-ray diffraction (XRD) spectroscopic methods including Fourier Transform Infrared spectroscopy, Raman Spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and Energy dispersive spectroscopy (EDS). We also characterized them using morphological techniques such as Field emission scanning electron microscopy (FE-SEM), and electrochemical approaches that included cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We examined thermal stability by thermogravimetric analysis (TG/DTA) and N2 adsorption-desorption isotherm by Brunauer-Emmett-Teller (BET) surface area method. Both materials were tested for the detection of lead (II) ions in aqueous media. Au nanoparticle incorporated CuBTC MOF showed great affinity and selectivity toward Pb2+ ions and achieved a lower detection limit (LOD) of 1 nM/L by differential pulse voltammetry (DPV) technique, which is far below than MCL for Pb2+ ions (0.03 µM/L) suggested by the United States (U.S.) Environmental Protection Agency (EPA) drinking water regulations.

Structural basis of host protein hijacking in human T-cell leukemia virus integration.

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.

Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase.

Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last ß-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.

A C-terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly.

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

Crystal structure of the Rous sarcoma virus intasome.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

Multifunctional facets of retrovirus integrase.

The retrovirus integrase (IN) is responsible for integration of the reverse transcribed linear cDNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3' OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3' OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus (HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.

Critical appraisal of elvitegravir in the treatment of HIV-1/AIDS.

Human immunodeficiency virus type 1 (HIV-1) integrase inhibitors belong to a novel class of antiretroviral drugs with high potency and better tolerability. Elvitegravir (EVG) is the second integrase inhibitor approved by the US Food and Drug Administration when administered in combination with a novel pharmacoenhancer, cobicistat (COBI), and two nucleoside/nucleotide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF). This combination of drugs (EVG/COBI/FTC/TDF) developed and marketed by Gilead Sciences Inc. (Foster City, CA, USA) as STRIBILD(®), is the first integrase inhibitor-based single-tablet regimen administered once-daily. In the USA, it has been approved for use in antiretroviral treatment-naïve HIV-1 patients with estimated creatinine clearance of >70 mL/min. The Department of Health and Human Services has approved EVG/COBI/FTC/TDF as one of preferred first-line regimens for HIV-1 treatment. In Europe, the European Medicines Agency has approved STRIBILD in treatment-naïve patients as well as in patients having no resistant mutation to any of the antiviral agents contained in STRIBILD. Its availability as a fixed-dose combination and once-daily dosage makes the adherence highly likely. However, it also discounts the possibility of dosage adjustment if needed.

Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors.

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.

A possible role for the asymmetric C-terminal domain dimer of Rous sarcoma virus integrase in viral DNA binding.

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3' DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.

First-in-class small molecule inhibitors of the single-strand DNA cytosine deaminase APOBEC3G.

APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration.

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.

HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex.

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC(50) values of ∼20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of >200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤5% of input DNA). The formation of the ISD complex was not dependent on 3'OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.

Raltegravir in HIV-1 infection: Safety and Efficacy in Treatment-naïve Patients.

The hunt for a compound which inhibits the HIV-1 integrase had been painstakingly difficult. Integrase is essential for viral replication as it mediates the integration of the viral DNA genome into the host DNA resulting in the establishment of the permanent provirus. Persistent efforts have resulted in the discovery of Raltegravir (Isentress, MK-0518), the first integrase inhibitor approved by US Food and Drug Administration for the treatment in HIV-1 infected patients. Numerous clinical studies with raltegravir have found it to be safe and effective in treatment naïve as well as treatment experienced patients. Adverse events associated with raltegravir based therapy are milder compared to previously available regimens. Raltegravir is metabolized primarily via glucuronidation mediated by uridine diphosphate glucuronosyltransferase and has a favorable pharmacokinetics independent of age, gender, race, food, and drug-drug interactions. Within a short period of time of its introduction, raltegravir has been included as one of DHHS recommended preferred regimen for the treatment of HIV-1 infection in treatment naïve patients.

Physical trapping of HIV-1 synaptic complex by different structural classes of integrase strand transfer inhibitors.

Raltegravir is an FDA approved inhibitor directed against human immunodeficiency virus type 1 (HIV-1) integrase (IN). In this study, we investigated the mechanisms associated with multiple strand transfer inhibitors capable of inhibiting concerted integration by HIV-1 IN. The results show raltegravir, elvitegravir, MK-2048, RDS 1997, and RDS 2197 all appear to encompass a common inhibitory mechanism by modifying IN-viral DNA interactions. These structurally different inhibitors bind to and inactivate the synaptic complex, an intermediate in the concerted integration pathway in vitro. The inhibitors physically trap the synaptic complex, thereby preventing target DNA binding and thus concerted integration. The efficiency of a particular inhibitor to trap the synaptic complex observed on native agarose gels correlated with its potency for inhibiting the concerted integration reaction, defined by IC(50) values for each inhibitor. At low nanomolar concentrations (<50 nM), raltegravir displayed a time-dependent inhibition of concerted integration, a property associated with slow-binding inhibitors. Studies of raltegravir-resistant IN mutants N155H and Q148H without inhibitors demonstrated that their capacity to assemble the synaptic complex and promote concerted integration was similar to their reported virus replication capacities. The concerted integration activity of Q148H showed a higher cross-resistance to raltegravir than observed with N155H, providing evidence as to why the Q148H pathway with secondary mutations is the predominant pathway upon prolonged treatment. Notably, MK-2048 is equally potent against wild-type IN and raltegravir-resistant IN mutant N155H, suggesting this inhibitor may bind similarly within their drug-binding pockets.

Molecular Interactions between HIV-1 integrase and the two viral DNA ends within the synaptic complex that mediates concerted integration.

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3' OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5'-end labeled viral DNA ends in the synaptic complex (0.68+/-0.09) was significantly different from that observed in the strand transfer complex (0.07+/-0.02). The calculated distances were 46+/-3 A and 83+/-5 A, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to approximately 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (>120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.

Expression and functions of transmembrane mucin MUC13 in ovarian cancer.

MUC13, a transmembrane mucin, is normally expressed in gastrointestinal and airway epithelium. Its aberrant expression has been correlated with gastric colon and cancer. However, the expression and functions of MUC13 in ovarian cancer are unknown. In the present study, the expression profile and functions of MUC13 were analyzed to elucidate its potential role in ovarian cancer diagnosis and pathogenesis. A recently generated monoclonal antibody (clone PPZ0020) was used to determine the expression profile of MUC13 by immunohistochemistry using ovarian cancer tissue microarrays and 56 additional epithelial ovarian cancer (EOC) samples. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with the normal ovary/benign tissues. Among all ovarian cancer types, MUC13 expression was specifically present in EOC. For the functional analyses, a full-length MUC13 gene cloned in pcDNA3.1 was expressed in a MUC13 null ovarian cancer cell line, SKOV-3. Here, we show that the exogenous MUC13 expression induced morphologic changes, including scattering of cells. These changes were abrogated through c-Jun NH(2) kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (P < 0.05) increases in cell motility, proliferation, and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with up-regulation of HER2, p21-activated kinase 1, and p38 protein expression. Our findings show the aberrant expression of MUC13 in ovarian cancer and that its expression alters the cellular characteristics of SKOV-3 cells. This implies a significant role of MUC13 in ovarian cancer.

Biochemical and biophysical analyses of concerted (U5/U3) integration.

Retrovirus integrase (IN) integrates the viral linear DNA genome ( approximately 10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.