Cloning and Characterization of the Antiviral Activity of Feline Tetherin/BST-2

Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection

Development and Evaluation of a Simple Assay for Marburg Virus Detection Using a Reverse Transcription-Loop-Mediated Isothermal Amplification Method▿

Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 102 copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 104 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic

Establishment of Teth-CRFK cells and reduction of RD-114 production in Teth-CRFK cells.

<p>(A) The expression of human tetherin in Teth-CRFK cells was analyzed by Western blotting using anti-FLAG antibody. (B) CRFK, Teth-CRFK, and HeLa cells were stained with PE-labeled anti-CD317 antibody and analyzed by FACS. (C) CRFK and Teth-CRFK cells (1×106 cells/well) were cultured for 3 days. RD-114 released into the culture supernatant was quantified by real-time RT-PCR for its viral RNA using primers targeting the RD-114 pol region. Data represent means ± standard deviation of three independent experiments. (D) The productions of infectious RD-114 from CRFK and Teth-CRFK cells were quantified by LacZ marker rescue assay. Significance by Student's t test: **, P<0.01.</p

Importance of <i>N</i>-linked glycosylation for the antiviral activity.

<p>Both RD-114 vector (100 ng) and the expression vector for wild-type or mutant feline Tetherin/BST-2 (50, 100, or 200 ng) were cotransfected into 293T cells. Cells and viruses were collected at 48 h after transfection, and analyzed by Western blotting (A). The intensities of the bands for virus and cell-associated Gag were quantified using a Fuji LAS3000 imaging system (B). The control vector (virus p28^CA/virus p28^CA + cellular p28^CA + p68^Gag) was set to 100%. Histograms represent the averages from three independent experiments (± standard deviation of the mean).</p

Feline Tetherin/BST-2 is insensitive to antagonism by HIV-1 Vpu.

<p>Both RD-114 vector (100 ng) and the expression vector for human or feline Tetherin/BST-2 containing FLAG-tag (200 ng), with or without the expression vector for Vpu containing Myc-tag (1 µg), were cotransfected into 293T cells. Cells and viruses were collected at 48 h after transfection, and analyzed by Western blotting.</p

Induction of Tetherin/BST-2 and reduction of RD-114 particle release by IFN treatment of feline cells.

<p>CRFK cells were treated with 100 or 1,000 U/ml of IFN-α for 24 h. (A) feline Tetherin/BST-2 mRNA and 18S rRNA were quantified by real-time RT-PCR. The numbers of feline Tetherin/BST-2 mRNA copies were normalized to one copy of 18S rRNA. Histograms represent the averages from three independent experiments (± standard deviation of the mean). (B) RD-114 viral RNA in the supernatant from IFN-treated or untreated CRFK cells were quantified by real-time RT-PCR.</p

Comparison of the predicted amino acid sequences of cat, dog, pig, mouse, and human Tetherin/BST-2 homologs.

<p>Cat (CRFK, FL74, and QN10S cells), dog, pig, mouse, and human Tetherin/BST-2 sequences were analyzed and aligned using GENETYX ver. 8 (GENETYX, Tokyo, Japan). The predicted transmembrane domain is boxed. Three Cys residues in the extracellular domain, which are important for dimerization, are shown with a red background. Two putative glycosylation sites are shown with a blue background. Ser residues of the predicted cleavage site prior to addition of a GPI anchor are shown with a yellow background.</p