21 research outputs found
Thermodynamics of Rev–RNA Interactions in HIV‑1 Rev–RRE Assembly
The
HIV-1 protein Rev facilitates the nuclear export of intron-containing
viral mRNAs by recognizing a structured RNA site, the Rev-response-element
(RRE), contained in an intron. Rev assembles as a homo-oligomer on
the RRE using its α-helical arginine-rich-motif (ARM) for RNA
recognition. One unique feature of this assembly is the repeated use
of the ARM from individual Rev subunits to contact distinct parts
of the RRE in different binding modes. How the individual interactions
differ and how they contribute toward forming a functional complex
is poorly understood. Here we examine the thermodynamics of Rev–ARM
peptide binding to two sites, RRE stem IIB, the high-affinity site
that nucleates Rev assembly, and stem IA, a potential intermediate
site during assembly, using NMR spectroscopy and isothermal titration
calorimetry (ITC). NMR data indicate that the Rev–IIB complex
forms a stable interface, whereas the Rev–IA interface is highly
dynamic. ITC studies show that both interactions are enthalpy-driven,
with binding to IIB being 20–30 fold tighter than to IA. Salt-dependent
decreases in affinity were similar at both sites and predominantly
enthalpic in nature, reflecting the roles of electrostatic interactions
with arginines. However, the two interactions display strikingly different
partitioning between enthalpy and entropy components, correlating
well with the NMR observations. Our results illustrate how the variation
in binding modes to different RRE target sites may influence the stability
or order of Rev–RRE assembly and disassembly, and consequently
its function
SIRT5 catalytic activity is necessary to interact with Nsp14.
A. Cartoon representation of the protein structure of SIRT5 catalytic site in complex with cofactor NAD and succinylated lysine substrate (SuK), showing conserved residues mutated in panel B. B. Strep-tag affinity-purification and western blot after transfection of Sirt5-KD HEK293T cells with Nsp14-strep and SIRT5 catalytic mutants, showing that the interaction with Nsp14 is lost in several mutants. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with increasing concentrations of SIRT5 inhibitor Sirt5-i. High concentrations of Sirt5-i prevent the interaction. D. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with NAMPT FK866 inhibitor (low cellular NAD), FK866 and NMN, or NMN alone (high cellular NAD). SIRT5 binding strength correlated with NAD levels. E. Pan-acetylation, malonylation and succinylation in SIRT5-KD HEK293T total or Strep-purified proteins, after transfection with Nsp14-Strep, GFP-strep control and/or SIRT5. No specific lysine modifications could be detected. F. Summary of mass spectrometry experiments. Nsp14-strep proteins purified from SIRT5-KD HEK293T, with or without co-transfection with SIRT5, were analyzed by mass spectrometry. No acetylation, malonylation, succinylation or glutarylation modifications could be detected.</p
<i>SIRT5</i>-KO cells mount a stronger innate immune response.
RNA-seq analysis of WT and SIRT5-KO A549-ACE2 cells infected or mock-infected for 3 days with SARS-CoV-2 at MOI = 1. n = 4. A. Volcano plots showing differentially expressed genes between the different conditions. Highlighted genes display a q-value q1 (orange), or both (purple). Left panel: SIRT5-KO vs WT in mock-infected cells. Middle: Infected vs mock-infected WT cells. Right: Infected vs mock-infected SIRT5-KO cells. B. Normalized gene count of SARS-CoV-2. C-D. Unsupervised clustering of the 3221 genes differentially expressed between at least two of the four conditions (qC: heatmap of normalized expression. D. Z-scores of differentially expressed genes as grouped by clustering. Colored lines represent the quantification of an individual gene whereas solid black lines show the cluster Tukey boxplot. E. Enrichment analysis of biological gene sets in the identified gene clusters (C and D).</p
<i>SIRT5</i>-KO cells express a higher basal level of viral restriction factors.
A. Expression heatmap of interferon-stimulated genes and other restriction factors, showing that mock-infected SIRT5-KO cells express higher basal levels of restriction factors, and that antiviral responses are stronger in SIRT5-KO cells. Data show mean log2 fold-change, compared to mock-infected WT, and the q-value between mock-infected WT and SIRT5-KO cells. Only genes differentially expressed between at least two conditions were included in the analysis (qB. RT-qPCR confirmation of restriction factors upregulated in non-infected SIRT5-KO cells (n = 8). Data show fold-changes compared to WT levels after normalization with ACTIN. Data show mean and SEM between replicates. p-values after unpaired two-tailed t-test are shown and asterisks summarize the results. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.</p
SARS-CoV-2 Nsp14 interacts with human SIRT5.
A. Cartoon representation of the protein structure of Nsp14/Nsp10 (PDB 7N0B) and SIRT5 (PDB 3YIR) shows the Nsp14 N-terminal ExoN domain and C-terminal MTase domain. B. Affinity-purification of Nsp14-strep and co-purification of endogenous SIRT5 after transfection in HEK293T cells, as shown by western blot. C. Immunofluorescence of transfected Nsp14-Strep and endogenous SIRT5 in A549 cells. D. CETSA in HEK293T cells transfected with Nsp14-step and/or SIRT5, showing an increase in the stability of SIRT5 and Nsp14 by western blot. E. Western blot showing the absence of SIRT5 in SIRT5-KD HEK293T cells. F. Strep-tag affinity-purification or Flag-tag immunoprecipitation, followed by western blot, after transfection with Nsp14-strep, Nsp10-flag and SIRT5 expression constructs. SIRT5 does not interact with Nsp10. 0.5 μg of each construct or of empty control plasmids were transfected in SIRT5-KD HEK293T cells in a six-well plate. G. Strep-tag affinity-purification and western blot after transfection of Nsp14-strep, SIRT5 and increasing concentrations of Nsp10-tag indicate competitive binding of SIRT5 and Nsp10. 0.5 μg of Nsp14-strep and SIRT5 plasmid were used in a 6-well plate, with 0, 0.5, 1 or 2 μg of Nsp10-Flag.</p
SARS-CoV-2 Nsp14 interacts with human SIRT1.
A. Co-purification of endogenous sirtuins SIRT1, 2, 3, 6 and 7 after transfection of Nsp14-strep in HEK293T cells, as shown by western blot. Loading and purification controls are the same as in Fig 1B. B. Strep-tag affinity-purification and western blot after transfection of HEK293T cells with Nsp14-strep and SIRT1 WT and H355Y catalytic mutant, showing that the interaction with Nsp14 is lost in H355Y mutant. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 inhibitor Ex-527. High concentrations of Ex-527 prevent the interaction. D-E. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 specific activator SRT1720 (D) or non-specific activator resveratrol (E). Both drugs were cytotoxic at high concentrations and the apparent decrease in SIRT1 binding correlated with a similar decrease in the input lanes. F. In vitro desuccinylation activity of purified SIRT5 incubated with increasing concentrations of Nsp14, showing no effect. G. In vitro methyltransferase activity of purified Nsp14 incubated with increasing concentrations of SIRT5, showing no specific effect. Unmethylated GpppG cap-analog was used as a substrate.</p
Levels of viral restriction factors.
A. Principal component analysis of RNA-seq samples, showing that replicates are well separated based on knockout and infection status. B. Normalized gene count of interferon-stimulated genes and restriction factors, from Fig 7. (TIF)</p
Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G
<div><p>Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.</p> </div
UBE2F is required for activation of CRL5<sup>Vif-CBFß</sup><i>in vitro</i>.
<p><b>A,</b> Diagram of the ubiquitination protocol used in panel <b>B</b>. <b>B, </b><i>In vitro</i> ubiquitination of A3G by recombinant CRL5<sup>Vif-CBFß</sup> with UBE2R1 as ubiquitin conjugating enzyme requires UBE2F. Immunoblots of ubiquitination reactions containing myc-tagged A3G as the substrate show high-molecular weight polyubiquitin chains, require all protein components of the ubiquitin and NEDD8 activating systems and are only observed when UBE2F (lane 9) but not when UBE2M (lane 10) is used as NEDD8 conjugating enzyme. A3A is not susceptible to Vif and was used as a negative control. <b>C,</b> Coomassie-stained SDS-PAGE of NEDD8ylation “pulse” reaction indicates that under conditions used in panel <b>B</b> indicate CUL5 is completely NEDD8ylated by UBE2F; only a minor fraction (<5%) is NEDD8ylated by UBE2M. <b>D, </b><sup>15</sup>N-HSQC spectral overlays of RBX2<sub>RING</sub> in the presence and absence of ∼100 µM, unlabeled full-length UBE2M (top) or UBE2F (bottom).</p
The UBE2F loop insertion is required for efficient viral infectivity.
<p><b>A,</b> Single-cycle infectivity assay of HIV<sub>NL4-3</sub>ΔVif produced from HEK293T cells stably depleted for UBE2F (white bars), or non-silencing control shRNA (black bars) in the presence of transfected HIV<sub>NL4-3</sub>ΔVif (1 µg), A3G-V5 (500 ng), Vif-FLAG (100 ng), and increasing amounts of RNAi-immune wild-type or ΔLoop UBE2F-myc (0.2, 1, 3 or 10 ng), a catalytic mutant of UBE2F harboring a cysteine to alanine change at position 116 (10 ng), or empty vector control (10 ng). Mean and +-SD of duplicate experiments are graphed. <b>B,</b> Immunoblots of cell lysates and virus particles corresponding to panel <b>A</b>. A3G levels in cellular lysates and virus particles in UBE2F KD cells transfected with increasing amounts of RNAi-immune UBE2F (compare lanes 3–6, left and right), catalytic mutant UBE2F (C116A) or increasing amounts of UBE2F (ΔLoop) (lanes 1, 2, 7–10 left and right). To discern A3G levels in the virion in the shSCR lanes the immnoblots were exposed longer and non-specific bands from the protein ladder became apparent, as indicated by an asterisk.</p