Thomas M. Mariano

Identification and sequence of an accessory factor required for activation of the human interferon γ receptor

Abstract Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon γ (Hu-IFN-γ), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-γ receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-γ receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-γ receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-γ.

Interaction between the components of the interferon gamma receptor complex

Interferon γ (IFN-γ) signals through a multimeric receptor complex consisting of two different chains: the IFN-γ receptor binding subunit (IFN-γR, IFN-γR1), and a transmembrane accessory factor (AF-1, IFN-γR2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-γ receptor complex, we examined the function of the receptor components in signal transduction upon IFN-γ treatment. A specific IFN-γR2:IFN-γ cross-linked complex was observed in cells expressing both IFN-γR1 and IFN-γR2 indicating that IFN-γR2 (AF-1) interacts with IFN-γ and is closely associated with IFN-γR1. We show that the intracellular domain of IFN-γR2 is necessary for signaling. Cells coexpressing IFN-γR1 and truncated IFN-γR2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-γR1 alone were unresponsive to IFN-γ treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1α were activated, and IFN-γR1 was phosphorylated only in cells expressing both IFN-γR1 and IFN-γR2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-γR2.

Differential Responsiveness of a Splice Variant of the Human Type I Interferon Receptor to Interferons

Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (αYAC) respond to all type I human interferons including IFN-αA, IFN-β, and IFN-ω. The αYAC contains at least two genes encoding interferon-α receptor (IFN-αR) chains that are required for response to type I human interferons: Hu-IFN-αR1 and Hu-IFN-αR2. We previously isolated a splice variant of the Hu-IFN-αR1 chain designated Hu-IFN-αR1s. Chinese hamster ovary cells containing a disrupted αYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-αR1 and Hu-IFN-αR1s chains. With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-αR1s) and with the Hu-IFN-αR1 chains: Hu-IFN-αA and IFN-ω. Two other type I interferons, Hu-IFN-αB2 and Hu-IFN-αF, are capable of signaling through the Hu-IFN-αR1 chain only and cannot utilize the splice variant Hu-IFN-αR1s. Hu-IFN-αR1 and Hu-IFN-αR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene. These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-αR1 receptor chain, and that the splice variant chain (Hu-IFN-αR1s) is functional.

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope

ABSTRACT In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains☆

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.

UVB light suppresses nitric oxide production by murine keratinocytes and macrophages.

Nitric oxide is an important mediator of excessive cell growth and inflammation associated with many epidermal proliferative disorders. It is a highly reactive oxidant generated in keratinocytes and macrophages via the inducible form of the enzyme nitric oxide synthase (NOS2). In the present studies, we examined the effects of ultraviolet light (UVB, 2.5-25mJ/cm(2)) on interferon-gamma (IFN-gamma)-induced expression of NOS2 in these cells. Transient transfection assays using wild-type and mutant NOS2 promoter/luciferase reporter constructs showed that DNA binding of the transcription factors Stat1 and NF-kappaB was essential for optimal expression of the NOS2 gene. Whereas NF-kappaB was constitutively expressed in both cell types, Stat1 phosphorylation and nuclear binding activity were dependent upon IFN-gamma. UVB light, which is used therapeutically to treat inflammatory dermatosis, was found to suppress IFN-gamma-induced expression of NOS2 mRNA and protein, and nitric oxide production in both keratinocytes and macrophages. In macrophages, this was associated with complete inhibition of NF-kappaB nuclear binding activity and partial (approximately 20-25%) reduction of Stat1 activity. In keratinocytes, both responses were partially reduced at the highest doses of UVB light (15-25mJ/cm(2)). Whereas in macrophages UVB light suppressed NOS2 wild-type promoter-luciferase reporter activity, this activity was stimulated in keratinocytes. These data suggest that UVB light functions to suppress NOS2 gene expression in macrophages by inhibiting the activity of key regulatory transcription factors. In contrast, in keratinocytes, inhibition occurs downstream of NOS2 promoter activity.

Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

Background The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV. Methodology/Principle Findings Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested. Conclusions Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.

Correction: Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

[This corrects the article on p. e72205 in vol. 8.].

Identification of a Pyranocoumarin Photosensitizer that is a Potent Inhibitor of Keratinocyte Growth

Photosensitizers are used in the treatment of epidermal proliferation and differentiation disorders such as psoriasis and vitiligo. In these studies, a ring‐expanded carbon homolog of the linear psoralen (furo[3,2‐g]benzopyran‐7‐one) class of photosensitizers, 4,10‐dimethyl‐2H,8H‐benzo[1,2‐b:5,4‐b′]dipyran‐2‐one (NDH2476), was synthesized and analyzed for biological activity. Following activation by ultraviolet light (UVA, 320‐400 nm), NDH2476 was found to be a potent inhibitor of keratinocyte growth (IC50 = 9 nm). Similar derivatives methylated in the pyran ring, or containing a saturated pyran ring structure, were markedly less active or inactive as photosensitizers. NDH2476 was found to intercalate and damage DNA following UVA light treatment as determined by plasmid DNA unwinding and nicking experiments. Taken together, these data demonstrate that an intact furan ring in psoralen photosensitizers is not required for keratinocyte growth inhibition or DNA damage. Our findings that low nanomolar concentrations of a benzopyranone derivative were active as a photosensitizer indicates that this or a structurally related compound may be useful in the treatment of skin diseases involving aberrant epidermal cell growth and differentiation.