The Interferon-Inducible Mouse Apolipoprotein L9 and Prohibitins Cooperate to Restrict Theiler’s Virus Replication

RESEARCH ARTICLE The Interferon-Inducible Mouse Apolipoprotein L9 and Prohibitins Cooperate to Restrict Theiler’s Virus Replication Marguerite Kreit1, Didier Vertommen1, Laurent Gillet2, Thomas Michiels1* 1 Université catholique de Louvain, de Duve Institute, Brussels, Belgium, 2 Université de Liège, FARAH Research Center and Faculté de Médecine Vétérinaire, Liège, Belgium * Abstract OPEN ACCESS Citation: Kreit M, Vertommen D, Gillet L, Michiels T (2015) The Interferon-Inducible Mouse Apolipoprotein L9 and Prohibitins Cooperate to Restrict Theiler’s Virus Replication. PLoS ONE 10(7): e0133190. doi:10.1371/journal.pone.0133190 Editor: Robert Shin Fujinami, University of Utah, UNITED STATES Received: October 15, 2014 Accepted: June 23, 2015 Published: July 21, 2015 Copyright: © 2015 Kreit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Apolipoprotein L9b (Apol9b) is an interferon-stimulated gene (ISG) that has antiviral activity and is weakly expressed in primary mouse neurons as compared to other cell types. Here, we show that both Apol9 isoforms (Apol9b and Apol9a) inhibit replication of Theiler’s murine encephalomyelitis virus (TMEV) but not replication of vesicular stomatitis virus (VSV), Murid herpesvirus-4 (MuHV-4), or infection by a lentiviral vector. Apol9 genes are strongly expressed in mouse liver and, to a lesser extent, in pancreas, adipose tissue and intestine. Their expression is increased by type I interferon and viral infection. In contrast to genuine apolipoproteins that are involved in lipid transport, ApoL9 has an intracytoplasmic localization and does not seem to be secreted. The cytoplasmic localization of ApoL9 is in line with the observation that ApoL9 inhibits the replication step of TMEV infection. In contrast to human ApoL6, ApoL9 did not sensitize cells to apoptosis, in spite of the presence of a conserved putative BH3 domain, required for antiviral activity. ApoL9a and b isoforms interact with cellular prohibitin 1 (Phb1) and prohibitin 2 (Phb2) and this interaction might contribute to ApoL9 antiviral activity. Knocking down Phb2 slightly increased TMEV replication, irrespective of ApoL9 overexpression. The antiviral activity of prohibitins against TMEV contrasts with the pro-viral activity of prohibitins observed for VSV and reported previously for Dengue 2 (DENV-2), Chikungunya (CHIKV) and influenza H5N1 viruses. ApoL9 is thus an example of ISG displaying a narrow antiviral range, which likely acts in complex with prohibitins to restrict TMEV replication. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: MK was the recipient of a FRIA fellowship from the belgian Fund for Scientific research and was further supported by a fellowship from the Faculty of Medicine (Univ. of Louvain) and by the following programs which also funded research: the DIANE convention of the belgian walloon region, the ARC funding of the (Communauté Française de Belgique) and the Interuniversitary Attraction Poles programme initiated by the Belgian Science Policy Office (IAP-P7/ 45 BELVIR). Introduction Type I interferons (IFNs) mediate their antiviral effects through the expression of IFN-stimulated genes (ISGs). Recent studies based on large-scale gene knock down and overexpression screenings have evaluated the antiviral activity of hundreds of ISGs acting against RNA and DNA viruses [1–4]. Some ISG products display direct antiviral activity and sometimes act on a narrow virus range. Others act by regulating signal transduction pathways controlling IFN PLOS ONE | DOI:10.1371/journal.pone.0133190 July 21, 2015 1 / 22 Antiviral Activity of ApoL9 and Prohibitins Competing Interests: The authors have declared that no competing interests exist. production and IFN responses and thus act on a broad range of viruses. The emerging picture is that a given virus is controlled by a specific range of ISGs, some of these ISGs being virus- or virus family-specific and others acting in a more general fashion. We recently identified a group of mouse ISG that are not or weakly expressed in primary neurons after IFN-α/β treatment. Among these genes was the gene encoding apolipoprotein 9b (Apol9b). Apolipoproteins are typically associated with the transport of lipids in the organism. Accordingly, human apolipoprotein L1 (ApoL1) was originally described as a member of the high density lipoprotein family, which is involved in cholesterol transport [5]. However, the other members of the ApoL family were classified on the basis of sequence homology to ApoL1 but their functions may have diverged and remain to be characterized. The ApoL family is highly conserved across species [6]. APOL genes have been implicated in diseases such as schizophrenia and osteoarthritis, and are upregulated by both type I and type II IFNs [6, 7]. In human, six ApoL-coding genes (APOL1, APOL2, APOL3, APOL4, APOL5 and APOL6) are clustered on chromosome 22q12 [8]. ApoL1 has been extensively studied and acts as a restriction factor for trypanosome infection [9]. In addition, ApoL1 and other human ApoL members (ApoL1, L2, L3 and L6) have emerged in high-throughput screenings of ISG activity, as proteins with antiviral activity against various classes of RNA viruses [1, 4]. ApoL1 restricted infection of cells by the AR86 strain of Sindbis virus and more modestly by Venezuelan equine encephalitis virus and human parainfluenza virus type 3, but increased infection by Yellow fever virus. ApoL2 slightly inhibited hepatitis C virus replication but had pro-viral activity toward Influenza A virus and respiratory syncytial virus (RSV). ApoL6 was reported to have antiviral activity against two picornaviruses, coxsackie B virus and poliovirus. ApoL6 also displays inhibitory activity against RSV [1, 4]. Overexpression of ApoL6 triggers apoptosis, which suggests that the antiviral effect of ApoL proteins may correlate with cell sensitization to apoptosis [10]. Mouse ApoL9b is a 310 amino acid-long protein, identical by 97% to ApoL9a. ApoL9a and ApoL9b (referred to collectively as ApoL9) are encoded by distinct genes. The murine Apolipoprotein L family is encoded by 12 genes and 1 pseudogene (Apol6, Apol7a, Apol7b, Apol7c, Apol7e, Apol8, Apol9a, Apol9b, Apol10a, Apol10b, Apol11a, Apol11b and pseudogene Apol10c) clustered on chromosome 15 (Fig 1). Murine ApoL6 is orthologous to human ApoL6, murine ApoL7a to human ApoL5, murine ApoL7b to human ApoL4 and murine ApoL8 to human ApoL2. It is unclear which human protein is orthologous to mouse ApoL9. We previously reported that ApoL9b is an antiviral ISG active against Theiler’s murine encephalomyelitis virus (TMEV or Theiler’s virus) [11]. The weak expression of Apol9 in IFN-treated mouse primary neurons contributes to t (...truncated)