ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

Peer reviewe

Kaposi's sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

Kaposi's sarcoma herpesvirus (KSHV) is an angiogenesis-inducing oncovirus whose ability to usurp the oxygen-sensing machinery is central to its oncogenicity. By upregulating the hypoxia-inducible factors (HIFs), KSHV reprograms infected cells to a hypoxia-like state, triggering angiogenesis. Here we identify a link between KSHV replicative biology and oncogenicity by showing that KSHV's ability to regulate HIF2α levels and localization to the endoplasmic reticulum (ER) in normoxia enables translation of viral lytic mRNAs through the HIF2α-regulated eIF4E2 translation-initiation complex. This mechanism of translation in infected cells is critical for lytic protein synthesis and contributes to KSHV-induced PDGFRA activation and VEGF secretion. Thus, KSHV regulation of the oxygen-sensing machinery allows virally infected cells to initiate translation via the mTOR-dependent eIF4E1 or the HIF2α-dependent, mTOR-independent, eIF4E2. This “translation initiation plasticity” (TRIP) is an oncoviral strategy used to optimize viral protein expression that links molecular strategies of viral replication to angiogenicity and oncogenesis.Fil: Méndez Solís, Omayra. University of Miami; Estados UnidosFil: Bendjennat, Mourad. University of Miami; Estados UnidosFil: Naipauer, Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. University of Miami; Estados UnidosFil: Theodoridis, Phaedra R.. University of Miami; Estados UnidosFil: Ho, J.J. David. University of Miami; Estados UnidosFil: Verdun, Ramiro E.. University of Miami; Estados UnidosFil: Hare, Joshua M.. University of Miami; Estados UnidosFil: Cesarman, Ethel. Weill Cornell Medicine; Estados UnidosFil: Lee, Stephen. University of Miami; Estados UnidosFil: Mesri, Enrique Alfredo. University of Miami; Estados Unido

Fluorescent Protein Inserts in between NC and SP2 Are Tolerated for Assembly, Release and Maturation of HIV with Limited Infectivity

We report the design of a fluorescent HIV construct that is labeled by insertion of fluorescent protein between the nucleocapsid (NC) and spacer peptide 2 (SP2) domains of Gag and further show that the fluorescent protein is released from its confines within Gag during maturation. This fluorescent HIV is capable of budding and maturation with similar efficiency to the parental virus. Virions generated using this design within the R8 HIV backbone pseudotyped with VSV-G were capable of delivering small RNA genomes encoding GFP to the target cells; however, the same design within the NL4-3 backbone has limited HIV infectivity. The virions generated by these constructs are approximately 165 ± 35 nm in size, which is significantly larger than wild type HIV. We suggest that this design has the potential to be a vehicle for protein and small guide RNA delivery

The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding.

HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding "on-time" and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release

Simulation of Gag.Pol release kinetics.

<p><b>(A)</b> Simulated curves (solid) fitting the experimental data (scatter) are shown for WT, ΔYP and ΔPTAP conditions of Gag.Pol PRwt and Gag.Pol PRΔ. Histograms show the distribution of delay times for each condition. <b>(B)</b> The number of Pol proteins attached to Gag (Blue) and cleaved but confined in the VLP (Red) are shown for three separate single VLP simulations.</p

Gag p6 alteration delays Gag VLP release.

<p>Kinetics of VLP release by Gag in U2OS cells with either p6 wild type or inactivated as indicated, western blot kinetics are shown where 200 ng of each Gag construct was used for transfection; both Cells and VLPs were collected at 1 hour intervals and immunoprobed using p24 antibody (left panels), Single cell imaging 12 hours post-transfection of mCherry fused Gag constructs as indicated was performed using TIRF microscopy, images were captured before and after cell detachment to visualize released VLPs (right panels). All experiments were performed 3 times with similar results.</p

Gag cargo size dictates the requirement for p6.

<p><b>(A)</b> The size of cargo fused to Gag was artificially modulated using tandem GFP proteins. Kinetics of VLP release in 293T cells of Gag with various number of tandem GFPs (n = 0, 1, 2 and 3) is shown. 200 ng of each Gag construct were used for transfection, and samples were collected at 8 hours intervals for 24 hours post-transfection. Release of Gag shows increased sensitivity to YP and PTAP mutations in presence of larger cargo sizes. <b>(B)</b> Densitometry values of the panels on (A) which correspond to the ratio of p24 in VLPs/Cells. All experiments were performed 3 times with similar results.</p

Gag VLP release can bypass ESCRT-I/ALIX for recruitment of ESCRT-III/VPS4.

<p><b>(A)</b> ESCRT-III/VPS4 is retained within released VLPs independently of ESCRT-I/ALIX recruitment. The ESCRT proteins, Tsg101, ALIX, CHMP4b, and VPS4A, were co-expressed as HA-tagged ORFs along with the Gag variants in 293T cells as indicated. Their retention in released VLPs indicates their recruitment during VLP budding. <b>(B)</b> Expression of dominant negative VPS4 inhibits VLP release by Gag. 293T cells were transfected two times successively at 24 hours interval with HA tagged VPS4 either wild type (WT) or dominant negative E228Q (EQ) then with the Gag variants as indicated. Cells and VLPs were collected 24 hours post-Gag transfection. All experiments were performed 3 times with similar results.</p

Gag cargo size is dependent on p6 for VLP release.

<p><b>(A)</b> Rescue of VLP release from Gag-cargo with p6 mutations through expression of Gag p6 variants. 200 ng of each construct were used to transfect 293T cells; samples were collected 24 hours post-transfection. <b>(B)</b> Expression in 293T cells of natural cargo (Pol) truncations, depicted on the schematic representation (top panel), reproduced the same molecular phenotype as the artificial cargo (GFP) in terms of strict requirement of p6 for efficient VLP release (bottom panels). 250 ng of each Gag construct were used for transfection; samples were collected 24 hours post-transfection. All experiments were performed 3 times with similar results.</p