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

In mammals, 2'-O-methylation of RNA is a molecular signature by which the cellular innate immune system distinguishes endogenous from exogenous messenger RNA1-3. However, the molecular functions of RNA 2'-O-methylation are not well understood. Here we have purified TAR RNA-binding protein (TRBP) and its interacting partners and identified a DICER-independent TRBP complex containing FTSJ3, a putative 2'-O-methyltransferase (2'O-MTase). In vitro and ex vivo experiments show that FTSJ3 is a 2'O-MTase that is recruited to HIV RNA through TRBP. Using RiboMethSeq analysis4, we identified predominantly FTSJ3-dependent 2'-O-methylations at specific residues on the viral genome. HIV-1 viruses produced in FTSJ3 knockdown cells show reduced 2'-O-methylation and trigger expression of type 1 interferons (IFNs) in human dendritic cells through the RNA sensor MDA5. This induction of IFN-α and IFN-ß leads to a reduction in HIV expression. We have identified an unexpected mechanism used by HIV-1 to evade innate immune recognition: the recruitment of the TRBP-FTSJ3 complex to viral RNA and its 2'-O-methylation.

Endogenous TRIM5α Function Is Regulated by SUMOylation and Nuclear Sequestration for Efficient Innate Sensing in Dendritic Cells.

During retroviral infection, viral capsids are subject to restriction by the cellular factor TRIM5α. Here, we show that dendritic cells (DCs) derived from human and non-human primate species lack efficient TRIM5α-mediated retroviral restriction. In DCs, endogenous TRIM5α accumulates in nuclear bodies (NB) that partly co-localize with Cajal bodies in a SUMOylation-dependent manner. Nuclear sequestration of TRIM5α allowed potent induction of type I interferon (IFN) responses during infection, mediated by sensing of reverse transcribed DNA by cGAS. Overexpression of TRIM5α or treatment with the SUMOylation inhibitor ginkgolic acid (GA) resulted in enforced cytoplasmic TRIM5α expression and restored efficient viral restriction but abrogated type I IFN production following infection. Our results suggest that there is an evolutionary trade-off specific to DCs in which restriction is minimized to maximize sensing. TRIM5α regulation via SUMOylation-dependent nuclear sequestration adds to our understanding of how restriction factors are regulated.

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

BACKGROUND: 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 ca. 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. RESULTS: 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. CONCLUSION: 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.

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

Implication of TRIM alpha and TRIMCyp in interferon-induced anti-retroviral restriction activities.

BACKGROUND: TRIM5 alpha is a restriction factor that interferes with retroviral infections in a species-specific manner in primate cells. Although TRIM5 alpha 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 TRIM5 alpha or TRIMCyp in IFN-induced anti-retroviral activities. RESULTS: First, we show that human type I IFN can enhance TRIM5 alpha expression in human, African green monkey and macaque cells, as well as TRIMCyp expression in owl monkey cells. In TRIM5 alpha-expressing primate cell lines, type I IFN has little or no effect on HIV-1 infection, whereas it potentiates 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 TRIM5 alpha 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. CONCLUSION: Together, our observations indicate that both TRIM5 alpha 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.