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

Kreit M, Vertommen D, Gillet L, Michiels T - PLoS ONE (2015)

Bottom Line: In contrast to genuine apolipoproteins that are involved in lipid transport, ApoL9 has an intracytoplasmic localization and does not seem to be secreted.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.ApoL9 is thus an example of ISG displaying a narrow antiviral range, which likely acts in complex with prohibitins to restrict TMEV replication.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, de Duve Institute, Brussels, Belgium.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

ApoL9 has antiviral activity against a restricted virus range.L929 cells were transduced with the TM942 vector expressing mCherry alone (Vector/ white columns), or with the MK85 (light grey columns) and MK44 (dark grey columns) derivatives of this vector, that express Apol9a and Apol9b respectively. Three days after transduction, cells were infected with the indicated GFP-expressing viruses. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells. A. Relative infection efficiency (mean and SD), as determined by flow cytometry, for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–8.5 hpi; KJ26: 0.5–10hpi; VSV-GFP: 4–7hpi; MuHV-4: 4–18hpi. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells and analyzed 2 days after transduction. Values were pooled from three independent experiments performed each with triplicate infection samples (n = 9). B. Infectious virus titers (mean and SD) measured by plaque assay for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–20 hpi; KJ26: 0.5–20hpi; VSV-GFP: 4–20hpi; MuHV-4: 4–28hpi. Virus titers were normalized to those generated in cells transduced with the empty vector (n = 4 for cells transduced with MK44 or infected with KJ26. n = 8 for other conditions). *: significance of the difference between cells expressing ApoL9 and cells carrying the empty vector.
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pone.0133190.g004: ApoL9 has antiviral activity against a restricted virus range.L929 cells were transduced with the TM942 vector expressing mCherry alone (Vector/ white columns), or with the MK85 (light grey columns) and MK44 (dark grey columns) derivatives of this vector, that express Apol9a and Apol9b respectively. Three days after transduction, cells were infected with the indicated GFP-expressing viruses. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells. A. Relative infection efficiency (mean and SD), as determined by flow cytometry, for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–8.5 hpi; KJ26: 0.5–10hpi; VSV-GFP: 4–7hpi; MuHV-4: 4–18hpi. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells and analyzed 2 days after transduction. Values were pooled from three independent experiments performed each with triplicate infection samples (n = 9). B. Infectious virus titers (mean and SD) measured by plaque assay for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–20 hpi; KJ26: 0.5–20hpi; VSV-GFP: 4–20hpi; MuHV-4: 4–28hpi. Virus titers were normalized to those generated in cells transduced with the empty vector (n = 4 for cells transduced with MK44 or infected with KJ26. n = 8 for other conditions). *: significance of the difference between cells expressing ApoL9 and cells carrying the empty vector.

Mentions: Antiviral activity of ApoL9 was examined, by flow cytometry, using GFP-expressing viruses representative of different virus families: i) KJ7 and KJ26, which are derived from persistent and neurovirulent Theiler’s virus strains respectively (Picornaviridae, positive-stranded RNA viruses); ii) vesicular stomatitis virus (VSV) (Rhabdoviridae, negative-stranded RNA virus); iii) murid herpesvirus-4 (MuHV-4)(Herpesviridae, DNA virus); and iv) TM944, a lentiviral vector derived from HIV (Retroviridae, RNA/DNA virus). L929 cells were first transduced with lentiviral vectors pMK85 and pMK44 co-expressing Apol9a or Apol9b and mCherry, or with the control vector, pTM942, expressing mCherry alone. Three days after transduction, cells were infected with the various GFP-expressing viruses and analyzed by flow cytometry. Replication of the various viruses (GFP fluorescence) was analyzed in the population of transduced (mCherry-positive) cells. As shown in Fig 4A, Apol9a and Apol9b expression significantly inhibited infection by both persistent and neurovirulent TMEV strains but had little impact, if any, on the other tested viruses. To confirm these results, infectious virus yield was examined by plaque assay. Therefore, cells were transduced with the same Apol9-expressing lentiviral vectors. Three days after transduction, mCherry-positive cells were sorted and cultured for 2 days before infection with the GFP-expressing TMEV, MuHV-4 and VSV derivatives. Infectious virus yield was measured by plaque assay (Fig 4B). Again, ApoL9 expression significantly reduced TMEV but not MuHV-4 or VSV production. These data confirm the restricted virus range of ApoL9 antiviral activity.


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

Kreit M, Vertommen D, Gillet L, Michiels T - PLoS ONE (2015)

ApoL9 has antiviral activity against a restricted virus range.L929 cells were transduced with the TM942 vector expressing mCherry alone (Vector/ white columns), or with the MK85 (light grey columns) and MK44 (dark grey columns) derivatives of this vector, that express Apol9a and Apol9b respectively. Three days after transduction, cells were infected with the indicated GFP-expressing viruses. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells. A. Relative infection efficiency (mean and SD), as determined by flow cytometry, for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–8.5 hpi; KJ26: 0.5–10hpi; VSV-GFP: 4–7hpi; MuHV-4: 4–18hpi. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells and analyzed 2 days after transduction. Values were pooled from three independent experiments performed each with triplicate infection samples (n = 9). B. Infectious virus titers (mean and SD) measured by plaque assay for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–20 hpi; KJ26: 0.5–20hpi; VSV-GFP: 4–20hpi; MuHV-4: 4–28hpi. Virus titers were normalized to those generated in cells transduced with the empty vector (n = 4 for cells transduced with MK44 or infected with KJ26. n = 8 for other conditions). *: significance of the difference between cells expressing ApoL9 and cells carrying the empty vector.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4510265&req=5

pone.0133190.g004: ApoL9 has antiviral activity against a restricted virus range.L929 cells were transduced with the TM942 vector expressing mCherry alone (Vector/ white columns), or with the MK85 (light grey columns) and MK44 (dark grey columns) derivatives of this vector, that express Apol9a and Apol9b respectively. Three days after transduction, cells were infected with the indicated GFP-expressing viruses. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells. A. Relative infection efficiency (mean and SD), as determined by flow cytometry, for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–8.5 hpi; KJ26: 0.5–10hpi; VSV-GFP: 4–7hpi; MuHV-4: 4–18hpi. Titer of the TM944 GFP-expression lentiviral vector was adjusted to transduce about 30% of cells and analyzed 2 days after transduction. Values were pooled from three independent experiments performed each with triplicate infection samples (n = 9). B. Infectious virus titers (mean and SD) measured by plaque assay for indicated GFP-expressing viruses. Infection conditions (MOI and time) were: KJ7: 0.25–20 hpi; KJ26: 0.5–20hpi; VSV-GFP: 4–20hpi; MuHV-4: 4–28hpi. Virus titers were normalized to those generated in cells transduced with the empty vector (n = 4 for cells transduced with MK44 or infected with KJ26. n = 8 for other conditions). *: significance of the difference between cells expressing ApoL9 and cells carrying the empty vector.
Mentions: Antiviral activity of ApoL9 was examined, by flow cytometry, using GFP-expressing viruses representative of different virus families: i) KJ7 and KJ26, which are derived from persistent and neurovirulent Theiler’s virus strains respectively (Picornaviridae, positive-stranded RNA viruses); ii) vesicular stomatitis virus (VSV) (Rhabdoviridae, negative-stranded RNA virus); iii) murid herpesvirus-4 (MuHV-4)(Herpesviridae, DNA virus); and iv) TM944, a lentiviral vector derived from HIV (Retroviridae, RNA/DNA virus). L929 cells were first transduced with lentiviral vectors pMK85 and pMK44 co-expressing Apol9a or Apol9b and mCherry, or with the control vector, pTM942, expressing mCherry alone. Three days after transduction, cells were infected with the various GFP-expressing viruses and analyzed by flow cytometry. Replication of the various viruses (GFP fluorescence) was analyzed in the population of transduced (mCherry-positive) cells. As shown in Fig 4A, Apol9a and Apol9b expression significantly inhibited infection by both persistent and neurovirulent TMEV strains but had little impact, if any, on the other tested viruses. To confirm these results, infectious virus yield was examined by plaque assay. Therefore, cells were transduced with the same Apol9-expressing lentiviral vectors. Three days after transduction, mCherry-positive cells were sorted and cultured for 2 days before infection with the GFP-expressing TMEV, MuHV-4 and VSV derivatives. Infectious virus yield was measured by plaque assay (Fig 4B). Again, ApoL9 expression significantly reduced TMEV but not MuHV-4 or VSV production. These data confirm the restricted virus range of ApoL9 antiviral activity.

Bottom Line: In contrast to genuine apolipoproteins that are involved in lipid transport, ApoL9 has an intracytoplasmic localization and does not seem to be secreted.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.ApoL9 is thus an example of ISG displaying a narrow antiviral range, which likely acts in complex with prohibitins to restrict TMEV replication.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, de Duve Institute, Brussels, Belgium.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus