Biomedical subjects

Scott Long

Publications and source records attributed to Scott Long.

6 recordsLinked to original sources

PubMed2006

Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II.

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

Capsid Proteins↗

PubMed2006

Identification and expression analysis of interferon gamma genes in channel catfish.

Multiple species of type I interferon (IFN) were recently identified in catfish (CF) (Ictalurus punctatus). Herein we extend these studies and report the existence of two distinct type II IFN genes in channel CF. As with zebrafish and the green spotted pufferfish, the two CF IFN-gamma genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-gamma2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-gamma genes contain the hallmarks of authentic IFN-gamma including: (1) a conserved nuclear localization site at the C terminus (CF IFN-gamma2 only), (2) an IFN-gamma signature sequence, (3) six putative helical regions within the mature protein, (4) one or more potential glycosylation sites, and (5) multiple mRNA instability motifs within the 3' untranslated region. Moreover, well-characterized CF T and NK cell clones were shown to synthesize IFN-gamma transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-gamma and is consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production. Collectively, these studies indicate that Siluriformes possess two evolutionarily conserved IFN-gamma genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.

Amino Acid Sequence↗

PubMed2006

Identification and expression analysis of cDNAs encoding channel catfish type I interferons.

Previously a cDNA encoding a putative interferon gene, designated CF IFN-1, was identified from a catfish EST library. However, its constitutive expression, absence of a signal peptide, and apparently low level of biological activity suggested that this cDNA likely encoded an expressed pseudogene. Since Southern blot analysis suggested the presence of two to three IFN genes, additional cDNAs were generated from catfish fibroblast and lymphoid cell lines using primers designed to conserved regions of zebrafish and catfish interferon. Using this approach, three novel CF IFN genes, two of which likely encode functional interferon molecules, were identified. At the amino acid level, similarity among CF IFNs ranged from 71% to 82%, whereas similarity to other fish IFNs ranged from 15% to 35%. Although CF IFN-3, like CF IFN-1, lacks a signal peptide, CF IFN-2 and -4 appear to encode full-length, signal sequence-bearing genes. Consistent with their putative identification as functional genes, CF IFN-2 and -4 were not expressed in unstimulated cell lines, and CF IFN-2 was rapidly upregulated in CCO cells in response to virus infection or treatment with dsRNA. Moreover, as with salmon, fugu, and zebrafish interferon genes, CF IFN-1 contained four introns whose locations were conserved not only with respect to other fish IFNs, but also with respect to mammalian IFN-lambda. While it is likely that CF IFNs represent Type I IFNs, several characteristics preclude assigning these cytokines to any particular subfamily.

Amino Acid Sequence↗

PubMed2004

Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex.

To elucidate cytolytic mechanisms in the channel catfish, lysates from catfish lymphoid and fibroblast cell lines were screened by Western blot analysis using a panel of antibodies reactive with components of the mammalian apoptotic pathway. Strong reactivity with three proteins (approximate Mr 70,000, 37,000, and 15,000) was seen using an antibody targeted to mammalian Fas ligand (FasL). The sizes of the two smaller proteins are consistent with their tentative designation as membrane-bound (37,000 Mr) and soluble (15,000 Mr) FasL. Treatments known to induce FasL in mammalian systems (e.g., PMA/calcium ionophore, UV-irradiation) induced expression of the 37,000- Mr protein in catfish T-cell lines. Moreover, expression of the 37,000- Mr protein in clonal T cells was up-regulated by increasing cell density. At the nucleotide level, homologues of Fas receptor (FasR), FADD, and caspase 8 were identified and characterized. These gene products likely constitute the teleost equivalent of the death-inducing signaling complex (DISC). FADD was constitutively expressed in all (T, B, macrophage, and fibroblast) cell lines examined as well as in peripheral blood lymphocytes (PBL), whereas FasR and caspase 8 were expressed in all cell lines except CCO, a FasL-positive fibroblast line. In contrast to FasL, expression of FasR and caspase 8 was inversely proportional to cell density. Collectively these studies identified four membrane-proximal proteins involved in the initiation of apoptosis in channel catfish and suggest that mechanisms of cell-mediated cytotoxicity in teleosts are similar to those used by mammals.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Identification of a cDNA encoding channel catfish interferon.

Despite considerable advances in our understanding of teleost immunity, relatively few cytokine genes, including those for interferon (IFN), have been identified at the molecular level. In contrast, numerous studies have shown that following virus infection or exposure to double-stranded RNA, fish or fish cells produce a soluble factor that is functionally similar to mammalian IFN. A putative catfish (CF) IFN cDNA was identified by BLASTX screening of a catfish EST library generated from a mixed lymphocyte culture enriched for NK-like cells. Consistent with its designation as a putative cytokine cDNA, the 3' non-translated region contained multiple copies of an RNA instability motif. Analysis of the deduced amino acid sequence of CF IFN showed low levels of identity/similarity to a panel of mammalian and avian IFN proteins, and markedly higher similarity to a recently identified zebrafish IFN. To determine if the identified cDNA encoded CF IFN, expression was monitored following infection of channel catfish ovary (CCO) cells with UV-inactivated catfish reovirus or exposure to double-stranded RNA, treatments which induce IFN or IFN-like activity in catfish and other species. In both cases, upregulation of putative CF IFN mRNA was detected. Moreover, upregulation of CF IFN mRNA was accompanied by the appearance of an antiviral factor in the culture medium. To confirm these results, recombinant CF IFN was synthesized in COS-7 cells and shown to have antiviral activity in CCO cells. Collectively, these results argue strongly that the identified catfish cDNA is an IFN homolog.

Amino Acid Sequence↗

PubMed2003

Induction of apoptosis in frog virus 3-infected cells.

The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins.

Amino Acid Chloromethyl Ketones↗