The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNß induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.

Functional Genomics Reveals Linkers Critical for Influenza Virus Polymerase.

UNLABELLED: Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis. IMPORTANCE: The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.

Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection.

UNLABELLED: Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses. IMPORTANCE: Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.

Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation.

Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae family) in complex with single-stranded RNA refined to 2.78 Å resolution as well as a 2.68 Å resolution structure of LEAV N-ssDNA complex. LEAV N is made up of an N- and a C-terminal lobe, with the RNA binding site located at the junction of these lobes. The LEAV N tetramer binds a 44-nucleotide-long single-stranded RNA chain. Hence, oligomerization of N is essential for encapsidation of the entire genome and is accomplished by using extensions at the N and C terminus. Molecular details of the oligomerization of N are illustrated in the structure where a circular ring-like tertiary assembly of a tetramer of LEAV N is observed tethering the RNA in a positively charged cavity running along the inner edge. Hydrogen bonds between N and the C2 hydroxyl group of ribose sugar explain the specificity of LEAV N for RNA over DNA. In addition, base-specific hydrogen bonds suggest that some regions of RNA bind N more tightly than others. Hinge movements around F20 and V125 assist in the reversal of capsidation during transcription and replication of the virus. Electron microscopic images of the ribonucleoprotein complexes of LEAV N reveal a filamentous assembly similar to those found in phleboviruses.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Henipavirus receptor usage and tropism.

Nipah (NiV) and Hendra (HeV) viruses are the deadliest human pathogens within the Paramyxoviridae family, which include human and animal pathogens of global biomedical importance. NiV and HeV infections cause respiratory and encephalitic illness with high mortality rates in humans. Henipaviruses (HNV) are the only Paramyxoviruses classified as biosafety level 4 (BSL4) pathogens due to their extreme pathogenicity, potential for bioterrorism, and lack of licensed vaccines and therapeutics. HNV use ephrin-B2 and ephrin-B3, highly conserved proteins, as viral entry receptors. This likely accounts for their unusually broad species tropism, and also provides opportunities to study how receptor usage, cellular tropism, and end-organ pathology relates to the pathobiology of HNV infections. The clinical and pathologic manifestations of NiV and HeV virus infections are reviewed in the chapters by Wong et al. and Geisbert et al. in this issue. Here, we will review the biology of the HNV receptors, and how receptor usage relates to HNV cell tropism in vitro and in vivo.

Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers.

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post-translational modifications such as covalent conjugation of ubiquitin and small ubiquitin-related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co-opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO-regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50) of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Generation of a Live Attenuated Influenza Vaccine that Elicits Broad Protection in Mice and Ferrets.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of T cells from W7-791-immunized mice conferred heterologous protection, indicating a role for T cell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones.

Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates.

BACKGROUND: The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template. The mechanism of this template commitment is unknown. Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates. RESULTS: RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles. Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined. duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E. coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions. Access of the RNA substrate to the enzyme within the viral cores was confirmed. CONCLUSIONS: These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template. Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity.

Dual expression of lymphoid/basophil markers on single blast cells transformed from chronic myeloid leukemia.

A 27-yr-old man developed blastic crisis after the chronic phase of Philadelphia chromosome positive chronic myeloid leukemia (CML). The blast cells expressed terminal deoxynucleotidyl transferase (TdT)+/common acute lymphoblastic leukemia antigen (CALLA)+ phenotypes, corresponding to common ALL type. A vincristine plus prednisolone regimen initially suppressed the blastic proliferation, but the blasts soon reappeared as lymphoblasts, and 65% of them possessed basophil-like granules. Immunologic markers were not altered. The blasts were negative for myeloperoxidase, Sudan black B and periodic acid-Schiff reactions, but were positive for toluidine blue (TB) stain and supravital peroxidase (PO) stain using diaminobenzidine (DAB). These blasts were considered to have immature basophil granules. The supravital staining, for TB or PO in combination with fluorescinated-CALLA staining, directly revealed that single blasts expressed both basophil and lymphoid markers. This biphenotypic blast population was found to be a distinct clone from the initial crisis clone by cytogenetic examination. These findings suggest that the CML clone is derived from a multipotent stem cell common to lymphoid and myeloid lineages, or that dual markers may be expressed on transformed lymphoid or basophil clone as the result of differentiation infidelity probably determined by the genetic derangement in acute crisis.

Prognostic significance of the morphological dysplastic changes in chronic myelogenous leukemia.

Various morphological dysplastic changes were observed in patients with chronic myelogenous leukemia, especially in the acute crisis. To clarify their significance, we divided 45 patients in the acute crisis into two groups by our scoring system, the dysplastic group and the non-dysplastic group. Five of 25 subjects in the non-dysplastic group entered complete remission. None of 20 subjects in the dysplastic group did so, and the mean survival after the onset of acute crisis is significantly shorter in the dysplastic group than in the non-dysplastic group. Some patients in the dysplastic group had obvious dysplastic changes several months before the acute crisis. These findings suggest that acute crises in some cases may occur with or be preceded by the development of dysplastic clones similar to myelodysplastic syndrome; these patients respond poorly to conventional chemotherapy.

Chromosomal abnormalities in lymphoid crisis of chronic myelogenous leukemia.

The karyotypes in six patients with Ph-positive chronic myelogenous leukemia (CML) were investigated during the lymphoid crisis associated with high levels of terminal deoxynucleotidyl transferase (TdT) and/or the common acute lymphoblastic antigen (CALLA). Five of the six patients had only the Ph chromosome, with no other karyotypic abnormalities. The remaining one patient had a hypodiploid karyotype: 44,XY, -1, + der(1;?)(p22;?), -3, -4, -6, -7, -8, -9, +22q-, + mar1, + mar2, + mar3. In four patients with lymphoid crisis expressing TdT and CALLA, the response to treatment with vincristine (VCR) and prednisolone (PRD) was satisfactory, except for the one patient whose karyotype was hypodiploid. A discussion is presented as to whether or not there is a correlation between the karyotypic changes, using banding methods, and TdT expression in patients whose blast cells were categorized morphologically as lymphoblastic at the onset of the blastic phase of CML. Sequential chromosome examinations during the chronic and blastic phases of CML were also performed in this study.

Chromosomal characteristics of chronic and blastic phases of Ph-positive chronic myeloid leukemia.

To evaluate the appearance of chromosome changes, in addition to the Philadelphia (Ph) chromosome, as predictive and diagnostic parameters of transformation in chronic myeloid leukemia (CML), such changes were analyzed in the chronic phase (CP) and compared with those of the blastic phase (BP) of CML. The common chromosome changes observed in the CP were loss of a Y (-Y), trisomy 8 (+8), an isochromosome for the long arm of chromosome #17 [i(17q)], a double Ph (+Ph), reciprocal translocations, and partial deletions. In most patients with chromosome changes in addition to the Ph, the percentage of abnormal clones increased steadily during the CP and was accompanied by other chromosome changes shortly before or at the onset of the BP, except for cases with -Y or i(17q) clones. In general, most chromosome changes observed shortly before or at the BP were complex. These facts suggest that complex chromosome changes could be utilized as predictive and diagnostic parameters of blastic transformation in CML.

Erythrocyte glutathione reductase activity and acquired trisomy #8 in various hematologic disorders.

The gene for erythrocyte glutathione reductase (E-GR) activity has been assigned to chromosome #8. In the present series, we examined the E-GR activity in 14 cases with chronic myelodysplastic syndrome (CMS, preleukemia), atypical acute myelogenous leukemia (AML), or chronic myelogenous leukemia (CML), with and without acquired trisomy #8. No difference in the incidence of high levels of this enzyme was found between two groups, i.e., those with and without trisomy #8 suggesting the existence of a complex regulatory system in addition to chromosome #8.

Isochromosome 17q in a case of myelofibrosis with myeloid metaplasia terminating in blastic transformation.

A case of myelofibrosis with myeloid metaplasia in a 61-year-old female patient is reported. Cytogenetic studies were performed using short-term culture without phytohemagglutinin. A chromosomal aberration of an isochromosome 17q, [i(17q)], was revealed in 88% of the metaphases of peripheral blood cells in the blastic phase. However, all metaphases of bone marrow cells in the chronic phase showed a normal karyotype. Furthermore, i(17q) was also observed in 10% of the metaphases of spleen cells examined 8 months before blastic transformation. In this case, therefore, the cells with i(17q) were associated with an abnormal clone of blastic transformation, with the abnormal clone originating in the spleen with myeloid metaplasia.

Cytogenetic studies of human T-cell leukemia virus type I carriers. A family study.

Recently, the chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL), and this anomaly has also been confirmed in the preleukemic state of adult T-cell leukemia (pre-ATL) patients. Because the cytogenetic abnormality at the stage of human T-cell leukemia virus type I (HTLV-I) carrier remains uncertain, we performed cytogenetic studies of lymphocytes stimulated with phytohemagglutinin in three HTLV-I carriers and three non-HTLV-I carriers in an ATL family. As a result, in three HTLV-I carriers, four of 311 cells examined (1.3%) had chromosome 14q11 anomaly. However, in three non-HTLV-I carriers, none of 260 cells examined had chromosome 14q11 anomaly. These results suggest that chromosome 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.

Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer.

The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oligonucleotides has indicated no apparent species- or ligand-dependent, nucleotide-specific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dependent, TCDD responsiveness in each of these species.