Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates <em>in vitro</em>

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

The Scripps Research Institute

Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link to the ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth. We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids were aquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.

Whales originated from aquatic artiodactyls in the Eocene epoch of India.

Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing coronavirus DMV assembly.

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles.

Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.

Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases.

The Eocene greenhouse climate state has been linked to a more vigorous hydrologic cycle at mid- and high latitudes; similar information on precipitation levels at low latitudes is, however, limited. Oxygen isotopic fluxes track moisture fluxes and, thus, the δ(18)O values of ocean surface waters can provide insight into hydrologic cycle changes. The offset between tropical δ(18)O values from sampled Eocene sirenian tooth enamel and modern surface waters is greater than the expected 1.0 per mil increase due to increased continental ice volume. This increased offset could result from suppression of surface-water δ(18)O values by a tropical, annual moisture balance substantially wetter than that of today. Results from an atmospheric general circulation model support this interpretation and suggest that Eocene low latitudes were extremely wet.

Latitudinal gradients in greenhouse seawater δ(18) O: evidence from Eocene sirenian tooth enamel.

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.

Sr/Ca and Ba/Ca ratios of bone are commonly used as biochemical indicators of trophic level in modern and fossil mammals. Concerns over the effects of diagenesis on Sr/Ca and Ba/Ca ratios of bone led archaeologists and paleontologists to favor tooth enamel, which is less prone to alteration. Sr/Ca and Ba/Ca ratios of bone, enamel, and dentin from three farm-raised steers (Bos taurus) and five wild white-tailed deer (Odocoileus virginianus) from central Missouri were compared. Our results show that changes in diet, discrimination, and growth rate during ontogeny can lead to significant differences in Sr/Ca and Ba/Ca ratios of different bioapatite types as well as significant differences within the same bioapatite forming at different times. Early- and late-forming tooth enamel can have significant differences in Sr/Ca and Ba/Ca ratios equivalent to almost one full trophic step. Although differences between early- and late-forming dentin are typically not significant, dentin Sr/Ca and Ba/Ca ratios are significantly greater than enamel values. This difference in Sr/Ca or Ba/Ca ratios between enamel and dentin from the same tooth can be greater than one full trophic step. These results have profound implications for the use of dental bioapatites in trophic level reconstructions. They highlight the importance of consistency in bioapatite selection, tooth selection, and relative location of sampling within the enamel cap. Furthermore, this expected difference in Sr/Ca and Ba/Ca ratios could be used as another means of checking for diagenetic alteration in ancient samples.

Ontogenetic variations in Sr/Ca and Ba/Ca ratios of dental bioapatites from Bos taurus and Odocoileus virginianus.

The human immunodeficiency virus type 1 (HIV) Tat protein, a potent activator of HIV gene expression, is essential for integrated viral genome expression and represents a potential antiviral target. Tat binds the 5'-terminal region of HIV mRNA's stem-bulge-loop structure, the transactivation-responsive (TAR) element, to activate transcription. We find that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid from a marine sponge, inhibits Tat-mediated transactivation of the integrated provirus by binding specifically to the TAR-binding domain of Tat. Working at subnanomolar concentrations, dCA reduces Tat-mediated transcriptional initiation/elongation from the viral promoter to inhibit HIV-1 and HIV-2 replication in acutely and chronically infected cells. Importantly, dCA abrogates spontaneous viral particle release from CD4(+)T cells from virally suppressed subjects on highly active antiretroviral therapy (HAART). Thus, dCA defines a unique class of anti-HIV drugs that may inhibit viral production from stable reservoirs and reduce residual viremia during HAART.

An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription.

Climate strongly affects the production of sediment from mountain catchments as well as its transport and deposition within adjacent sedimentary basins. However, identifying climatic influences on basin stratigraphy is complicated by nonlinearities, feedback loops, lag times, buffering and convergence among processes within the sediment routeing system. The Palaeocene/Eocene thermal maximum (PETM) arguably represents the most abrupt and dramatic instance of global warming in the Cenozoic era and has been proposed to be a geologic analogue for anthropogenic climate change. Here we evaluate the fluvial response in western Colorado to the PETM. Concomitant with the carbon isotope excursion marking the PETM we document a basin-wide shift to thick, multistoried, sheets of sandstone characterized by variable channel dimensions, dominance of upper flow regime sedimentary structures, and prevalent crevasse splay deposits. This progradation of coarse-grained lithofacies matches model predictions for rapid increases in sediment flux and discharge, instigated by regional vegetation overturn and enhanced monsoon precipitation. Yet the change in fluvial deposition persisted long after the approximately 200,000-year-long PETM with its increased carbon dioxide levels in the atmosphere, emphasizing the strong role the protracted transmission of catchment responses to distant depositional systems has in constructing large-scale basin stratigraphy. Our results, combined with evidence for increased dissolved loads and terrestrial clay export to world oceans, indicate that the transient hyper-greenhouse climate of the PETM may represent a major geomorphic 'system-clearing event', involving a global mobilization of dissolved and solid sediment loads on Earth's surface.

Mark Clementz

Department of Infectious Diseases

The Scripps Research Institute

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Mark Clementz

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Infectious Diseases

The Scripps Research Institute

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Department of Infectious Diseases