Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities

<p>Abstract</p> <p>Background</p> <p>TRIM5α is a restriction factor that interferes with retroviral infections in a species-specific manner in primate cells. Although TRIM5α is constitutively expressed, its expression has been shown to be up-regulated by type I interferon (IFN). Among primates, a particular case exists in owl monkey cells, which express a fusion protein between TRIM5 and cyclophilin A, TRIMCyp, specifically interfering with HIV-1 infection. No studies have been conducted so far concerning the possible induction of TRIMCyp by IFN. We investigated the consequences of IFN treatment on retroviral restriction in diverse primate cells and evaluated the implication of TRIM5α or TRIMCyp in IFN-induced anti-retroviral activities.</p> <p>Results</p> <p>First, we show that human type I IFN can enhance TRIM5α expression in human, African green monkey and macaque cells, as well as TRIMCyp expression in owl monkey cells. In TRIM5α-expressing primate cell lines, type I IFN has little or no effect on HIV-1 infection, whereas it potentates restriction activity against N-MLV in human and African green monkey cells. In contrast, type I IFN treatment of owl monkey cells induces a great enhancement of HIV-1 restriction, as well as a strain-tropism independent restriction of MLV. We were able to demonstrate that TRIM5α is the main mediator of the IFN-induced activity against N-MLV in human and African green monkey cells, whereas TRIMCyp mediates the IFN-induced HIV-1 restriction enhancement in owl monkey cells. In contrast, the type I IFN-induced anti-MLV restriction in owl monkey cells is independent of TRIMCyp expression.</p> <p>Conclusion</p> <p>Together, our observations indicate that both TRIM5α and TRIMCyp are implicated in IFN-induced anti-retroviral response in primate cells. Furthermore, we found that type I IFN also induces a TRIMCyp-independent restriction activity specific to MLV in owl monkey cells.</p

Residual HIV-1 DNA Flap-independent nuclear import of cPPT/CTS double mutant viruses does not support spreading infection

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a <it>ca</it>. 100 bp strand displacement at the central termination sequence (CTS). The central DNA Flap is a determinant of HIV-1 nuclear import, however, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. Therefore, to determine whether or not the DNA Flap is essential for HIV-1 nuclear import, we generated double mutant (DM) viruses, combining cPPT and CTS mutations to abolish DNA Flap formation.</p> <p>Results</p> <p>The combination of cPPT and CTS mutations reduced the proportion of viruses forming the central DNA Flap at the end of reverse transcription and further decreased virus infectivity in one-cycle titration assays. The most affected DM viruses were unable to establish a spreading infection in the highly permissive MT4 cell line, nor in human primary peripheral blood mononuclear cells (PBMCs), indicating that the DNA Flap is required for virus replication. Surprisingly, we found that DM viruses still maintained residual nuclear import levels, amounting to 5-15% of wild-type virus, as assessed by viral DNA circle quantification. Alu-PCR quantification of integrated viral genome also indicated 5-10% residual integration levels compared to wild-type virus.</p> <p>Conclusion</p> <p>This work establishes that the central DNA Flap is required for HIV-1 spreading infection but points to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication.</p

TRBP recruits a 2’O-methyltranferase on Human Immunodeficiency Virus type 1 RNA : mechanism of innate immunity escape

TRBP (TAR RNA Binding Protein), est un facteur activateur de la réplication du Virus de l'Immunodéficience Humaine de type 1 (VIH-1). Cette protéine cellulaire qui interagit avec les ARN double brins est connue pour son rôle crucial dans la voie des miRNA. Isolée pour sa capacité à interagir avec la séquence leader TAR présente à l'extrémité 5' de tous les ARN du VIH-1, TRBP favorise la réplication du VIH-1 au niveau post-transcriptionnel, en partie via l'inhibition de la PKR (Protéine Kinase ARN dépendante).Dans le but de mieux comprendre les mécanismes moléculaires par lesquels TRBP facilite la réplication du VIH-1, le complexe protéique associé à TRBP a été purifié par immunoprécipitation par double affinité et identifié par spectrométrie de masse. En plus des facteurs déjà connus, un nouveau partenaire à activité ARN 2'-O-méthyltransférase (2'-OMTase) potentielle a été copurifié : la protéine FTSJ3. Chez les eucaryotes supérieurs, deux 2'-OMTases permettent la méthylation des ARNm cellulaires au niveau de la position ribose 2'-O- du premier (coiffe 1) et du deuxième nucléotide (coiffe 2). Cette coiffe 1/2 est une signature moléculaire permettant de discriminer les ARNm endogènes et exogènes. Dans la cellule, MDA5, un senseur cytoplasmique, reconnait les ARN exogènes non coiffés et déclenche la production d'interférons (IFNs) de type I pour établir un état antiviral. Pour échapper à la réponse immune innée, certains virus ont développé des mécanismes leur permettant de mimer une coiffe 1/2.Le VIH ne code pas pour une activité 2'-OMTase. Cependant FTSJ3, de par son interaction avec TRBP, se retrouve à proximité de l'extrémité 5' de l'ARN viral. Cette 2'-OMTase méthyle l'ARN TAR in vitro, qui, transfecté dans les cellules monocytaires humaines U937 n'induit plus la production d'IFNs de type I. A l'inverse, le virus VIH-1 produit en l'absence de FTSJ3 déclenche une induction de l'expression des IFNs de type I dépendante de MDA5 dans les cellules U937. L'expression de ce virus est atténuée suite à un défaut d'import nucléaire. Ainsi, ces travaux montrent que la protéine FTSJ3, recrutée au niveau de l'extrémité 5' de l'ARN du VIH-1 par TRBP, facilite la réplication du VIH-1 en assurant la synthèse d'une coiffe 1/2 qui permet au VIH-1 d'échapper à la reconnaissance par le senseur MDA5 et à l'induction des IFNs de type I. Cette étude met en évidence un nouveau mécanisme permettant au VIH-1 d'échapper à la détection par le système d'immunité innée cellulaire.TRBP (TAR RNA Binding Protein) is a cellular RNA binding protein that facilitates the replication of Human Immunodeficiency Virus type 1 (HIV-1). Isolated for its ability to bind HIV-1 TAR sequence present at the 5' end of all HIV-1 RNA, TRBP promotes HIV-1 replication at a post-transcriptional level by counteracting the antiviral activity of the protein kinase R (PKR).To gain more insight on how TRBP enhances HIV-1 replication, TRBP associated factors were purified using tandem immunoaffinity purification and identified by mass spectrometry. In addition to already known associated factors, a new protein with a putative RNA 2'-O-methyltransferase activity (2'OMTases) was copurified: FTSJ3. In higher eukaryotes, cellular mRNA are methylated on 2'-O ribose position on the first (Cap 1) and second nucleotide (Cap 2). This capping provides a molecular signature for the discrimination of endogenous versus exogenous mRNA. In the cell, MDA5, a cytoplasmic sensor, recognizes exogenous uncapped RNA and activate type I interferons (IFNs) production to establish an antiviral state. To evade innate immune response, some viruses have evolved mechanisms to mimics cap 1/2.HIV-1 does not encode a 2'O-MTase activity. However, owing to its interaction with TRBP, FTSJ3 is recruited at the 5' end of the viral genome and methylates TAR RNA in vitro. When capped by FTSJ3, TAR does not induce type I IFNs anymore when transfected in monocytic cell line U937. Conversely, HIV-1 viruses produced in FTSJ3 knock-down cells triggers type I IFNs expression through MDA5 sensing. This virus is attenuated, expressed in low amounts because of a block at the level of HIV-1 nuclear import. This study shows that FTSJ3 is recruited to HIV-1 5' end TAR sequence by TRBP and facilitates HIV-1 replication. HIV-1 RNA capping allows HIV-1 escape from MDA5 sensing and type I IFN induction. This study highlights a new way of HIV-1 escape from innate immune system

FTSJ3 is an RNA 2′-O-methyltransferase recruited by HIV to avoid innate immune sensing

International audienc

Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities-0

� or left untreated (-), and transduced 24 h later with VSV-pseudotyped GFP-expressing N-MLV (N), HIV-1 (HIV) or NB-MLV (NB) at low (MOI = 0.5) or high (MOI = 5) multiplicity of infection (as determined on MDTF cells). The percentage of transduced (GFP-positive) cells was determined by FACS 48 h post-transduction.<p><b>Copyright information:</b></p><p>Taken from "Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities"</p><p>http://www.retrovirology.com/content/5/1/59</p><p>Retrovirology 2008;5():59-59.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483995.</p><p></p

Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities-4

-MLV or NB-MLV at a MOI of 5. CsA (5 μM) was added 2 h before transduction. Percentage of transduced cells was determined by FACS 48 h post-transduction. Fold restriction represents the ratio of untreated to IFN-β-treated cells to be transduced by the GFP-encoding retroviral vectors. Results are representative of two independent experiments with comparable results. A significant enhancement of retroviral restriction is defined as a ratio > 2. . OMK cells were stimulated with IFN-β and transduced 24 h later with VSV-pseudotyped GFP-expressing HIV-1, SIVmac or HIV-1 SCA vectors at a MOI of 1. The percentage of transduced cells was determined by FACS 48 h post-transduction. Fold restriction represents the ratio of untreated/IFN-β-treated cells to be transduced. As in panel A, a significant enhancement of retroviral restriction is defined as a ratio > 2.<p><b>Copyright information:</b></p><p>Taken from "Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities"</p><p>http://www.retrovirology.com/content/5/1/59</p><p>Retrovirology 2008;5():59-59.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483995.</p><p></p

Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities-2

1, HA2 or A3), as indicated, and stimulated 24 h later with 1000 U/ml of IFN-β for 8 h. Total RNA was extracted and the levels of TRIM5α mRNA were determined by quantitative RT-PCR and normalized to GAPDH. The mean ± SD of duplicates is shown. . HeLa or Vero cells were transfected with siRNA targeting luciferase (Luc), TRIM5α(H1 or HA2) or TRIM5α(HA2 or A3), as indicated. The next day, cells were stimulated with 1000 U/ml of IFN-β for 8 h and challenged with a GFP-expressing N-MLV vector. The percentage of GFP-positive cells was determined by FACS 48 h post-transduction. Data are from a typical experiment representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities"</p><p>http://www.retrovirology.com/content/5/1/59</p><p>Retrovirology 2008;5():59-59.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483995.</p><p></p

Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities-1

GFP-expressing N-MLV (N), HIV-1 (HIV), NB-MLV (NB), B-MLV (B) or SIVmac (SIV) at low (MOI = 0.5) or high (MOI = 5) multiplicity of infection. The percentage of transduced cells was determined by FACS 48 h post-transduction.<p><b>Copyright information:</b></p><p>Taken from "Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities"</p><p>http://www.retrovirology.com/content/5/1/59</p><p>Retrovirology 2008;5():59-59.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483995.</p><p></p

Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities-3

as indicated, and stimulated 24 h later with 1000 U/ml of IFN-β for 8 h. Total RNA was extracted and the levels of TRIMCyp mRNA were determined by quantitative RT-PCR and normalized to GAPDH. The mean ± SD of duplicates is shown. . OMK cells were transfected with anti-Luc (diamonds) or anti-TRIMCyp (triangles) siRNA and transduced 48 h later with increasing doses of HIV-1. The percentage of GFP-positive cells was determined by FACS 48 h post-transduction. . Same experiment as in panel B, except that cells were challenged with HIV-1, N-MLV, B-MLV or NB-MLV (at a MOI of 5), following siRNA and IFN-β treatments. The percentage of GFP-positive cells was determined by FACS 48 h post-transduction. Data are from a typical experiment representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Implication of TRIMalpha and TRIMCyp in interferon-induced anti-retroviral restriction activities"</p><p>http://www.retrovirology.com/content/5/1/59</p><p>Retrovirology 2008;5():59-59.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483995.</p><p></p