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

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

Products of Gag.Pol processing by PR during VLP production.

<p>Among cleavage sites in Gag and Gag-Pol, the SP1/NC and SP2/p6 sites are the most rapidly cleaved by PR [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005657#ppat.1005657.ref067" target="_blank">67</a>]. If the neck closes under normal conditions (WT p6), soluble PR-containing fragments are trapped in VLPs and continue processing the remaining cleavage sites on Gag and Gag-Pol, which leads to release of mature virions. In the case of delayed neck closure, soluble PR-containing fragments diffuse to host cytosol and progeny virions are devoid of Pol products (ΔPTAP and to lesser extent ΔYP VLPs). PM, plasma membrane.</p