s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery.

The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.

Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus infecting salmonid fish. The virus is adapted to low temperature and has a replication optimum between 10-15 °C. In this study the subcellular localization and protein interactions for the protein encoded by the largest open reading frame of gene segment 8 (s8ORF2) were investigated. In ISAV infected cells the s8ORF2 protein was found mainly in the cytosol but a minor fraction of cells expressed the protein in the nucleus as well. Green fluorescent protein-tagged s8ORF2 did not leak out of the cell when the plasma membrane was permeabilized, suggesting interactions with intracellular structural components. The s8ORF2 protein exists both as monomer and homodimer, and co-immunoprecipitation experiments strongly suggests it binds to the ISAV fusion-, nucleo- and matrix proteins. Two versions of s8ORF2 were detected with apparent molecular weights of 24-26 and 35 kDa in lysates of infected cells. The 35 kDa type is an early viral protein while the smaller version appears during the later phases of infection. The 24-26 kDa type was also the predominant form in viral particles. The s8ORF2 protein has previously been shown to bind RNA and interfere with interferon induction and signaling. Here we found that a fraction of the s8ORF2 protein pool in infected cells is likely to be conjugated to the interferon stimulated gene 15 (ISG15) and ubiquitin. Furthermore, several endogenous proteins pulled down by the s8ORF2 protein were identified by liquid chromatography mass spectrometry (LC-MS).

A polyprotein-expressing salmonid alphavirus replicon induces modest protection in atlantic salmon (Salmo salar) against infectious pancreatic necrosis.

Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

Transcriptional regulation of gene expression of infectious salmon anaemia virus segment 7.

The nuclear replication and gene splicing of orthomyxoviruses are unique among RNA viruses. Segment 7 of infectious salmon anaemia virus (ISAV) is the only segment that undergoes splicing. Two proteins are encoded by this segment, the non-structural antagonist (ISAV-NS) of the innate immune response that is translated from the unspliced collinear transcript, and a nuclear exporting protein (ISAV-NEP) that is translated from the spliced mRNA. Here we report the transcription profiles for these ISAV proteins. The appearance of the spliced ISAV-NEP mRNA was delayed and the relative amount was less but slowly accumulated to 20-30% to that of the collinear NS mRNA. In cells transfected with segment 7 the ratio between spliced and collinear mRNA was approximately 10%. A highly conserved, possible structured RNA, in the region of the 3' splicing site of the segment is speculated as being important for the regulation of the efficiency of the splicing.

Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).

The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.

Infectious salmon anaemia virus nuclear export protein is encoded by a spliced gene product of genomic segment 7.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing anaemia and circulatory disease with high mortality in farmed Atlantic salmon (Salmo salar). Orthomyxoviruses are unusual as RNA viruses as they replicate in the nucleus and some viral transcripts undergo splicing. The nuclear replication necessitates a tightly controlled nuclear import and export of viral proteins. From ISAV genomic segment 7 two known mRNAs are transcribed; one collinear with the genomic segment, coding for the non-structural protein, and one spliced transcript, S7ORF2, coding for a protein with unknown function. Here we report initial functional analysis of the S7ORF2 protein. The results indicate that S7ORF2 protein gradually accumulates in the host cell during virus replication cycle, locates predominantly in the cytoplasm and is a part of purified virus particles. Trapping of S7ORF2 in the nucleus was obtained by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export, indicating that S7ORF2 use CRM1 for the nuclear exit. Immunofluorescent staining of cells over-expressing both S7ORF2 and matrix protein (M) showed co-localization in the nucleus. However, S7ORF2 protein was found to interact with both the viral nucleoprotein (NP) and M proteins in ISAV infected cells as well as in purified viral particles. These results indicate that the S7ORF2 could be called the ISAV nuclear export protein, ISAV/NEP.

Alternative translation initiation generates cytoplasmic sheep prion protein.

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein ((GFP)PrP) in N2a cells, with variable sequence context surrounding the start codon Met(1). (GFP)PrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of (GFP)PrP were detected intracellularly, starting in frame from Met(17). When (GFP)PrP was expressed with a compromised Kozak sequence ((GFP)PrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of (GFP)PrP*, whereas the N-terminal fragments starting from Met(17) were still present. Formation of these N-terminal fragments was completely abolished when Met(17) was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met(17) is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.

Proteolytic processing of the ovine prion protein in cell cultures.

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.

Estrogenic effects of environmental chemicals: an interspecies comparison.

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17beta-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER approximately rtER approximately rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.

Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7.

Several antiepileptic drugs (AEDs) are associated with anti-cancer activity. At the same time, many AEDs alter endocrine function with phenytoin (PHT) and phenobarbital (PB) causing-reduced free fractions of sex-steroid hormones, while VPA induces hyperandrogenism. Changes in sex-steroid hormone levels are known to affect apoptosis in endocrine tissue. The aim of the study was to investigate the influence of the antiepileptic drugs PHT, PB, VPA and lamotrigine (LTG) on estrogen-stimulated cell growth of human breast cancer cells (MCF-7), and to evaluate whether this effect could be related to a direct estrogen receptor (ER) binding. VPA reduced cell growth at therapeutically relevant concentrations; half-maximum effect of VPA on cell growth was 230 microM. PHT (100 microM) and PB (10 microM) reduced cell growth by 47 and 21%, respectively. None of the drugs had affinity to isolated estrogen receptors, and excess of estrogen was not able to abolish the growth inhibition provoked by VPA. However, sub-therapeutic concentrations of VPA (100 microM) mimicked estrogen by inducing cell growth (11%) in an estrogen-depleted medium, an effect that was abolished by adding an estrogen receptor antagonist. In conclusion; the estrogen receptor appear to be indirectly activated by sub-therapeutic concentrations of VPA, but therapeutic concentrations of VPA inhibits cell growth by mechanisms that do not seem to involve the estrogen receptor or estrogen stimulation.

Effects of the environmental oestrogens bisphenol A, tetrachlorobisphenol A, tetrabromobisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl on oestrogen receptor binding, cell proliferation and regulation of oestrogen sensitive proteins in the human breast cancer cell line MCF-7.

Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen-like potency using several end-points in MCF-7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro- and tetrabromo-bisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl all showed oestrogen-like properties in MCF-7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF-7 cells, although their EC50s were 1,000 to 80,000 times higher than the EC50 of 17beta-oestradiol. Bisphenol A and 4-hydroxybiphenyl induced cell growth in MCF-7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17beta-oestradiol. The other chemicals tested induced less than 50% of the maximum 17beta-oestradiol-stimulated cell growth. Bisphenol A, 4-hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen-regulated proteins, progesterone receptor and pS2, whereas 4,4'-dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17beta-oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF-7 cells. By measuring several oestrogen-dependent endpoints it seems that some xeno-oestrogens cause an imbalanced oestrogen-response. Their ability and potency in mimicking 17beta-oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen-linked parameter.

Brominated phenols: characterization of estrogen-like activity in the human breast cancer cell-line MCF-7.

A large number of halogenated phenols are detected in the blood of humans, fish and wild-animals. We have characterized the estrogen-like activity of phenol, 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and 4-tert-butylphenol (tert-BP) using the estrogen-dependent human breast cancer cell line MCF-7. 4-BP, 2,4-DBP and 4-tert-BP all bind to the estrogen receptor (ER) with approximately 10,000-fold less affinity than 17 beta-estradiol (17 beta-E). 2,4,6-TBP was only able to displace 43% of radiolabelled estrogen when tested at concentrations up to 1 microM, whereas phenol had no affinity for the ER. 4-tert-BP stimulated cell growth and induced estrogen-regulated proteins such as the progesterone receptor (PgR) and pS2. The brominated phenols, however, although binding to the ER, did not stimulate cell growth or increase the levels of the PgR or pS2, or reduce the level of 17 beta-E induced pS2. On the contrary, 4-BP, 2,4-DBP and partly 4-tert-BP reduced 17 beta-E-stimulated cell growth apparently by an ER independent mechanism.