Jacob S. Yount

Robust Fluorescent Detection of Protein Fatty-Acylation with Chemical Reporters

Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

S-Palmitoylation and Ubiquitination Differentially Regulate Interferon-induced Transmembrane Protein 3 (IFITM3)-mediated Resistance to Influenza Virus

Background: IFITM3 is a protein of the innate immune system that inhibits viral infections. Results: S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, whereas ubiquitination decreases endolysosome localization and antiviral activity. Conclusion: IFITM3 is dually and opposingly regulated by posttranslational modifications, and study of these modifications has led to an unpredicted intramembrane topology model. Significance: Understanding modes of IFITM3 regulation is critical for dissecting molecular mechanisms controlling viral inhibition. The interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular restriction factor that inhibits infection by influenza virus and many other pathogenic viruses. IFITM3 prevents endocytosed virus particles from accessing the host cytoplasm although little is known regarding its regulatory mechanisms. Here we demonstrate that IFITM3 localization to and antiviral remodeling of endolysosomes is differentially regulated by S-palmitoylation and lysine ubiquitination. Although S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, ubiquitination decreases localization with endolysosomes and decreases antiviral activity. Interestingly, autophagy reportedly induced by IFITM3 expression is also negatively regulated by ubiquitination. However, the canonical ATG5-dependent autophagy pathway is not required for IFITM3 activity, indicating that virus trafficking from endolysosomes to autophagosomes is not a prerequisite for influenza virus restriction. Our characterization of IFITM3 ubiquitination sites also challenges the dual-pass membrane topology predicted for this protein family. We thus evaluated topology by N-linked glycosylation site insertion and protein lipidation mapping in conjunction with cellular fractionation and fluorescence imaging. Based on these studies, we propose that IFITM3 is predominantly an intramembrane protein where both the N and C termini face the cytoplasm. In sum, by characterizing S-palmitoylation and ubiquitination of IFITM3, we have gained a better understanding of the trafficking, activity, and intramembrane topology of this important IFN-induced effector protein.

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

ABSTRACT Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. IMPORTANCE SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

Phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (IFITM3) dually regulates its endocytosis and ubiquitination.

Background: IFITM3 restricts the fusion of viruses within endolysosomes. Results: Phosphorylation of IFITM3 on Tyr20 blocks IFITM3 endocytosis and ubiquitination. Conclusion: Tyr20 of IFITM3 is part of a YXXΦ endocytosis signal and has a dual role in regulating IFITM3 ubiquitination. Significance: Identifying mechanisms regulating IFITM3 trafficking and activity is crucial for understanding and manipulating IFITM3 biology for combating virus infections. Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3.

Cytokine-Independent Upregulation of MDA5 in Viral Infection

ABSTRACT The RNA helicases RIG-I and MDA5 detect virus infection of dendritic cells (DCs) leading to cytokine induction. Maximal sensitivity for virus detection by these helicases is obtained after their upregulation, which is thought to occur primarily through type I interferon (IFN) signaling. Here we demonstrate that in response to paramyxovirus infection, RIG-I upregulation requires type I IFN whereas MDA5 expression is increased by Sendai virus infection independently of signaling mediated by type I IFN, STAT1, tumor necrosis factor alpha, or NF-κB. This MDA5 upregulation is largely lost in IRF3 knockout DCs and is achieved in type I IFN-deficient cells expressing constitutively active IRF3.

Palmitoylation on Conserved and Nonconserved Cysteines of Murine IFITM1 Regulates Its Stability and Anti-Influenza A Virus Activity

ABSTRACT The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.

Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation

Significance Cysteine fatty acylation (S-fatty acylation) regulates the stability, trafficking, and activity of proteins in eukaryotes. Current methods to study S-fatty acylation rely on the metabolic incorporation of fatty acid analogs or selective chemical labeling and affinity purification, which limits the detection of endogenous S-fatty acylated protein isoforms and levels. Here we demonstrate that the biochemical exchange of acyl groups on cysteines with defined mass-tags enables the direct visualization of endogenous S-fatty acylated protein levels. The application of this mass-tag labeling method revealed that site-specific S-fatty acylation of interferon-induced transmembrane protein 3 controls the antiviral activity of this IFN-induced effector. Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

To the Editor: Sterile alpha motif domain– and HD domain–containing protein 1 (SAMHD1) is a cellular dNTP triphosphohydrolase (dNTPase) that restricts HIV-1 replication in myeloid cells and resting CD4+ T cells by degrading dNTPs and limiting viral reverse transcription1–5. Purified recombinant SAMHD1 also has exonuclease activity when synthetic nucleic acids or HIV-1 gag and tat RNAs transcribed in vitro are used as substrates6. Ryoo et al.7 recently suggested that SAMHD1 restricts HIV-1 infection through its ribonuclease (RNase) activity by cleaving the viral RNA genome. By using SAMHD1 mutants purported to specifically retain dNTPase (SAMHD1Q548A) or RNase (SAMHD1D137N) activities, Ryoo et al.7,8 proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in nondividing cells. They also suggested that SAMHD1 phosphorylation at T592 negatively regulated its RNase activity7. To extend these findings7, we measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-dependent HIV-1 reverse transcription was bypassed. We co-transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phosphoablative, but dNTPase-active, mutant (SAMHD1T592A; refs. 9–11) into human embryonic kidney (HEK) 293T cells and assessed intracellular HIV-1 Gag protein synthesis and viral particle release in the supernatants. This transfection-based HIV-1 production is independent of reverse transcription requiring intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA–mediated gene expression. Intracellular HIV-1 Gag protein levels, p24 capsid levels in released HIV-1 virions and infectivity were not reduced by the ectopic expression of WT SAMHD1 or SAMHD1T592A mutant (Supplementary Fig. 1), which suggests that SAMHD1 cannot inhibit HIV-1 production after the reverse transcription step, regardless of its phosphorylation at T592. These findings are consistent with our previous results showing that the expression of WT SAMHD1 or of the SAMHD1T592A mutant in dividing cells does not restrict HIV-1 infection11,12. Our results suggest that SAMHD1 does not have broad nuclease activity, but do not rule out a specific nucleolytic interaction between SAMHD1 and incoming HIV-1 genomic RNA (gRNA). Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, we reproduced the key experiments of Ryoo et al.7. Their conclusion that SAMHD1 restricts HIV-1 through RNase activity was based on the differential activity of the SAMHD1 mutants, SAMHD1D137N and SAMHD1Q548A (ref. 7). We independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure that there were no other disabling mutations in the constructs. We then examined the HIV-1 restriction and intracellular dNTP regulation by these mutants and WT SAMHD1 by following the protocol of Ryoo et al.7. Our results show that both the SAMHD1D137N and SAMHD1Q548A mutants were expressed at similar levels to that of WT SAMHD1, and each efficiently restricted HIV-1 infection and decreased dATP, dGTP and dTTP levels in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (Fig. 1a–c). SAMHD1 expression did not significantly decrease dCTP levels, as compared to vector control cells (Fig. 1c, right), probably owing to the different biosynthesis pathway of dCTP. Notably, Ryoo et al.7 showed only dCTP levels, but not dATP, dGTP or dTTP levels, in SAMHD1expressing or control cells. Previous studies have used a SAMHD1D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation and HIV-1 restriction of SAMHD1 (refs. 13–15). The SAMHD1D137A mutant has no detectable dNTPase activity or HIV-1 restriction in vitro, owing to its inability to form a stable tetramer14,15. It is possible that the SAMHD1D137N mutant might be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. It is also possible that an in vitro dNTPase assay using purified recombinant SAMHD1 proteins might not fully reflect the dNTPase activity of SAMHD1 in PMA-differentiated U937 cells. However, these possibilities remain to be examined to explain how the SAMHD1D137N mutant restricts HIV-1 infection if its dNTPase activity is impaired. In contrast to the results of Ryoo et al.7, the SAMHD1D137N and SAMHD1Q548A mutants in our experiments do not have differing anti-HIV-1 activities, and neither lacks the ability to lower cellular dNTP levels. Ryoo et al.7 reported an approximately twofold decrease in HIV-1 gRNA levels in PMA-differentiated U937 cells expressing WT SAMHD1 or SAMHD1D137N, but not the SAMHD1Q548A mutant, as compared to the control cells at 3 h and 6 h postinfection (h.p.i.), which suggests SAMHD1-mediated HIV-1 gRNA degradation7. By contrast, we detected comparable levels of HIV-1 gRNA in PMAdifferentiated U937 cells expressing SAMHD1 (WT, SAMHD1D137N and SAMHD1Q548A mutants) and the vector control cells at 1, 3 and 6 h.p.i., respectively (Fig. 1d), showing that, in our study, SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 h.p.i, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that the expression of WT SAMHD1, SAMHD1D137N and SAMHD1Q548A mutants significantly reduced HIV-1 late reverse transcription products as compared to the vector control cells (Fig. 1e), correlating well with a reduced intracellular dNTP pool (Fig. 1c). Thus, in our study, these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction. Seamon et al.16 reported that trace exonuclease activities of recombiSAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

Sendai virus infection induces efficient adaptive immunity independently of type I interferons.

ABSTRACT Adaptive immunity in response to virus infection involves the generation of Th1 cells, cytotoxic T cells, and antibodies. This type of immune response is crucial for the clearance of virus infection and for long-term protection against reinfection. Type I interferons (IFNs), the primary innate cytokines that control virus growth and spreading, can influence various aspects of adaptive immunity. The development of antiviral immunity depends on many viral and cellular factors, and the extent to which type I IFNs contribute to the generation of adaptive immunity in response to a viral infection is controversial. Using two strains (Cantell and 52) of the murine respiratory Sendai virus (SeV) with differential abilities to induce type I IFN production from infected cells, together with type I IFN receptor-deficient mice, we examined the role of type I IFNs in the generation of adaptive immunity. Our results show that type I IFNs facilitate virus clearance and enhance the migration and maturation of dendritic cells after SeV infection in vivo; however, soon after infection, mice clear the virus from their lungs and efficiently generate cytotoxic T cells independently of type I IFN signaling. Furthermore, animals that are unresponsive to type I IFN develop long-term anti-SeV immunity, including CD8+ T cells and antibodies. Significantly, this memory response is able to protect mice against challenge with a lethal dose of virus. In conclusion, our results show that primary and secondary anti-SeV adaptive immunities are developed normally in the absence of type I IFN responsiveness.