SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.     

Relation of structural polarity to responses of cyclic 3', 5'-adenosine monophosphate accumulation and thyroid hormone release in cultured human thyroid follicles

The relationship of structural polarity to functional activities was examined in cultured human thyroid follicles, which were isolated from the thyroid gland of patients with Graves' disease by collagenase treatment. Structural polarity was examined morphologically by electron microscopy, while the functional response to bovine TSH was examined by measuring intracellular cAMP accumulation and T3 release. In freshly isolated thyroid follicles, structural polarity was normal and TSH induced significant cAMP accumulation but no significant release of T3. After culture for 5 days the structural polarity of thyroid follicles became inverted in the absence of thyroid stimulators, but normal polarity was retained in the presence of TSH or dibutyryl cAMP [Bu)2 cAMP). The response to TSH of cAMP accumulation increased markedly after culture in either the presence or absence of TSH, suggesting that cAMP accumulation in response to TSH is not related to structural polarity. In contrast, thyroid follicles cultured without thyroid stimulators showed no significant T3 release in response to TSH, whereas those cultured with TSH or (Bu)2 cAMP showed significant T3 release in response to TSH. These data indicate that in cultured human thyroid follicles, the responses to TSH of cAMP accumulation and T3 release are not always correlated. Among many other explanations, the results were at least compatible with the idea that normal structural polarity is necessary for thyroid hormone release in response to TSH.     

Characterization and function of Met150Gln mutant of copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013

The mutant (M150Q-NIR) replacing the Met150 ligand of the type 1 Cu center in Achromobacter cycloclastes nitrite reductase (AcNIR) with Gln has been physicochemically and functionally characterized. The electronic absorption and CD spectra of M150Q-NIR are similar to those of mavicyanin and stellacyanin having the 2His, Cys, and Gln ligands, but the EPR signal has an axial character, although their blue copper proteins show rhombic EPR signals. The mutant has about 80% catalytic activity of AcNIR. Moreover, the midpoint potential (E(1/2)) of M150Q-NIR is +113 mV vs. NHE at pH 7.0, being negatively shifted compared to that of AcNIR (+240 mV). Although the intermolecular electron-transfer process from Achromobacter cycloclastes pseudoazurin (pAz) to M150Q-NIR was not detected, the pAz mutant (M86Q-pAz) replacing the Met86 ligand with Gln transfers one electron to the NIR mutant with an intermolecular electron-transfer rate constant (k(ET)) of 2.3 x 10(5)M(-1)s(-1).     

Mechanical and chemical properties of cysteine-modified kinesin molecules.

To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome

The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.     

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.     

Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.