Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells.

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.

Characterization of the Interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19).

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Hantavirus nucleocapsid protein has distinct m7G cap- and RNA-binding sites.

Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that carry three negative stranded RNA molecules as their genome. Hantavirus nucleocapsid protein (N) is encoded by the smallest S segment genomic RNA (viral RNA). N specifically binds mRNA caps and requires four nucleotides adjacent to the cap for high affinity binding. We show that the N peptide has distinct cap- and RNA-binding sites that independently interact with mRNA cap and viral genomic RNA, respectively. In addition, N can simultaneously bind with both mRNA cap and vRNA. N undergoes distinct conformational changes after binding with either mRNA cap or vRNA or both mRNA cap and vRNA simultaneously. Hantavirus RNA-dependent RNA polymerase (RdRp) uses a capped RNA primer for transcription initiation. The capped RNA primer is generated from host cell mRNA by the cap-snatching mechanism and is supposed to anneal with the 3' terminus of vRNA template during transcription initiation by single G-C base pairing. We show that the capped RNA primer binds at the cap-binding site and induces a conformational change in N. The conformationally altered N with a capped primer loaded at the cap-binding site specifically binds the conserved 3' nine nucleotides of vRNA and assists the bound primer to anneal at the 3' terminus. We suggest that the cap-binding site of N, in conjunction with RdRp, plays a key role during the transcription and replication initiation of vRNA genome.

Metnase mediates chromosome decatenation in acute leukemia cells.

After DNA replication, sister chromatids must be untangled, or decatenated, before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation, causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint, and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain, and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance, Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro, purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus, Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors, and Metnase is a potential therapeutic target for small molecule interference.

Hippocampal N-methyl-D-aspartate receptor subunit expression profiles in a mouse model of prenatal alcohol exposure.

BACKGROUND: Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. METHODS: We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. RESULTS: We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. CONCLUSIONS: Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.