Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex

The 5′-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5′-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2′-O positions, catalyzed by nsp14 N7-MTase and nsp16 2′-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2′-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs

Identification and functional characterization of SlDronc in Spodoptera littoralis

Background Apoptosis is responsible for eliminating damaged and virus-infected cells, regulating normal cell turnover, and maintaining the immune system’s development and function. Caspases play a vital role in both mammal and invertebrate apoptosis. Spodoptera littoralis is a generalist insect herbivore that is one of the most destructive pests in tropical and subtropical areas and attacks a wide range of commercially important crops. Although S. littoralis is a model organism in the study of baculovirus infection, its apoptotic pathway has not been explored. Methods We cloned a new caspase gene named sldronc in S. littoralis using Rapid Amplification of cDNA Ends (RACE). We then measured caspase activity on synthetic caspase substrates and S. littoralis’ effector caspase. SlDronc’s function in the apoptotic pathway and its interaction with caspase inhibitors were also tested in SL2 cells. Results We found that the initiator caspase SlDronc cleaved and activated effector caspase in S. littoralis. SlDronc overexpression induced apoptosis in SL2 cells, and Sldronc knockdown decreased apoptosis induced by UV irradiation in SL2 cells. Our results indicate that SlDronc acts as an apoptotic initiator caspase in S. littoralis. Additionally, we found that processed forms of SlDronc increased in the presence of N-terminally truncated S. littoralis inhibitors of apoptosis (SlIAP) and that SlDronc was inhibited by P49. This study contributes to the further understanding of S. littoralis’ apoptotic pathway and may facilitate future studies on baculovirus infection-induced apoptosis

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells

The Strica Homolog AaCASPS16 Is Involved in Apoptosis in the Yellow Fever Vector, Aedes albopictus.

Caspases are a family of cysteine proteases playing essential roles during apoptosis. Seven caspases identified in Drosophila were Dronc, Dredd, Strica, Dcp-1, Decay, Drice and Damm. Among them, Strica is an insect-specific caspase containing a long serine- and threonine- rich prodomain, of which function is not yet well studied. Here we identified a homolog of strica from Aedes albopictus, named as Aacasps16. Aacasps16 encoded a protein containing a putative serine- and threonine-rich prodomain and a well conserved caspase catalytic domain. AaCASPS16 shared high identity with dipteran insects Strica homologs. Alignment showed that the closest relative of AaCASPS16 was Aedes aegypti AeCASPS16. The expression profiles of Aacasps16 during developmental and adult stages were analyzed. Purified recombinant AaCASPS16 exhibited the highest caspase activity to WEHD, which is the substrate preferred by human caspase-9. AaCASPS16 induced apoptosis when over-expressed in C6/36 cells. AaCASPS16 was processed during apoptosis induced by actinomycin D and ultraviolet irradiation treatment, whereas partial silencing of Aacasps16 reduced actinomycin D- and ultraviolet irradiation-triggered apoptosis in C6/36 cells. Taken together, our study identified AaCASPS16 as a novel apoptotic caspase in Aedes albopictus

Recombinant SfDredd possessed the strongest activity on effector caspase substrates.

<p>Wild type SfDredd and active site mutant SfDredd-C443A with C-terminal His-tag <b>(A)</b> or N-terminal His-tag <b>(B)</b> were incubated, respectively with 13 types of synthetic caspase substrates (20 μmol/L) and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. The data were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ***<i>P</i> < 0.001.</p

The protein level of SfDredd increased in ActD-treated Sf9 cells.

<p>Sf9 cells were treated with ActD at a final concentration of 250 ng/mL. <b>(A)</b> Cell pictures were taken 6 h post ActD treatment (magnification ×200). <b>(B)</b> Sf9 cells were harvested 6 h after ActD (250 ng/mL) treatment, and cell lysates were analyzed by immunoblotting using antibody against SfDredd.</p

Recombinant SfDronc possessed strongest activity on initiator caspase substrates.

<p>Wild type SfDronc and active site mutant SfDronc-C310A with C-terminal His-tag were incubated, respectively with 13 types of synthetic caspase substrates (20 μmol/L) and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. The data were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ***<i>P</i> < 0.001, *<i>P</i> < 0.05.</p

SfDredd underwent autocatalytic cleavage when expressed and purified from <i>E</i>. <i>coli</i>.

<p>C-terminally His-tagged <b>(A)</b> or N-terminally His tagged <b>(B)</b> SfDredd and their active site mutant SfDredd-C443A were expressed and purified from <i>E</i>. <i>coli</i> and detected by immunoblotting using antibody against His-tag following SDS-PAGE. Diagrams showing the full length SfDredd and cleaved subunits are on the right. A short vertical black line was used to indicate where lanes were removed and separate parts of the same Western blot image were joined together. His: His-tag, Pro: prodomain, LS: large subunit, L: linker, SS: small subunit.</p

Phylogenetic analysis of SfDredd with selected insect caspases.

<p>The predicted amino acid sequence of SfDredd together with 27 selected insect caspases were aligned and the phylogenetic tree was constructed by MEGA 5.05 using the neighbor-joining method. The sequences include the following: SfDredd, SfDronc and Sf-caspase-1 from <i>Spodoptera frugiperda</i>, Spli-caspase-5 and Spli-caspase-6 from <i>Spodoptera litura</i>, Se-caspase-5 and Se-caspase-6 from <i>Spodoptera exigua</i>, Hv-caspase-6 from <i>Heliothis virescens</i>, Ha-caspase-5 and Ha-caspase-6 from <i>Helicoverpa armigera</i>, Ms-caspase-6 from <i>Manduca sexta</i>, Gm-caspase-6 from <i>Galleria mellonella</i>, Bm-caspase-1, Bm-caspase-3/a (BmICE), Bm-caspase-4, Bm-caspase-5 (BmDronc) and Bm-caspase-6 (BmDredd) from <i>Bombyx mori</i>, Ld-caspase-5 from <i>Lymantria dispar</i>, Pr-caspase-5 from <i>Pieris rapae</i>, AeDredd and AeDronc from <i>Aedes aegypti</i>, Dcp1, DECAY, DAMM, DrICE, STRICA, DmDredd and DmDronc from <i>Drosophila melanogaster</i>. Genbank accession numbers of sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151016#pone.0151016.s004" target="_blank">S4 Table</a>. *: Bm-caspase-4 belongs to Lep-caspase-4 which is a peculiar caspase, whether it belongs to initiator or effector caspase has not been decided [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151016#pone.0151016.ref024" target="_blank">24</a>].</p

The sequence of SfDredd.

<p>The predicted amino acid sequence of SfDredd is shown in alignment with Dredd homologs from <i>Bombyx mori</i> (BmDredd), <i>Aedes aegypti</i> (AeDredd) and <i>Drosophila melanogaster</i> (DmDredd-PE). The amino acid residues identical among 4 Dredds are indicated by white letters within black boxes, the amino acid residues identical among 3 Dredds are indicated by white letters within dark gray boxes, and the amino acid residues identical between 2 Dredds are indicated by black letters within light gray boxes. The alignment was performed using ClustalX 2.1 and modified by GeneDoc 3.2. Secondary structures were predicted using JPred3. Box: catalytic center, closed circles: conserved residues responsible for the catalytic reaction, black arrow: predicted cleavage sites.</p