Calmodulin 2 Functions as an RNA Chaperone in Prokaryotic Cells

Plants express many calmodulin (CaM) isoforms. These proteins regulate the growth, development and environmental stress responses of plants by modulating targets. Herein, the Arabidopsis CaM2 isoform was found to be crucial for cold adaptation in prokaryotic cells, similar to the Escherichia coli cold shock protein CspA. Expressing CaM2 or CspA in the cold-sensitive E. coli BX04 mutant complemented the cold-sensitive phenotype under cold stress, but expression of CaM1, CaM7 or CML8 (CaM8) did not. Similar to RNA chaperones such as CspA, CaM2 strongly interacted with nucleic acids and its nucleic acid-binding capacity was much higher than that of CaM7, despite there being only a single amino acid difference between these two isoforms. Microscopic observation of CaM2-GFP revealed that CaM2 plays roles in both the nucleus and cytosol where RNA molecules are abundant. These results suggest that CaM2 can positively modulate cold stress responses by interacting with nucleic acid targets. Furthermore, CaM2 has both nucleic acid targets, similar to CaM7, and protein targets such as CAMTA3.     

Direct Interaction between Two Viral Proteins,the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C^ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C^ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C^ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C^ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C^ATPase is an essential component of the replication complex, and (iv) 2C^ATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.     

HdeB Functions as an Acid-protective Chaperone in Bacteria

Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.     

Ku70作为RNA解旋酶调节miR-124的加工成熟及神经细胞的分化

目的 Ku70蛋白主要通过其DNA结合特性参与双链DNA断裂（DSB）的非同源端连接（NHEJ）修复,有报道称其具有RNA结合功能,本文探索Ku70是否具有RNA解旋酶活性并影响miRNA加工成熟。方法 利用RNA免疫共沉淀（RIP）测序结合生物信息学分析Ku蛋白结合的RNA;蛋白质印迹法（Western blot,WB）结合定量反转录PCR（qRT-PCR）检测Ku蛋白与miRNAs的表达关系;生物膜干涉技术（BLI）实验分析Ku蛋白与RNA的结合能力;电泳迁移率变动分析（EMSA）实验确定Ku70及Ku80的RNA解旋酶活性;形态学检测结合WB分析Ku70调节miR-124引起的神经细胞功能变化;免疫荧光结合形态学分析寨卡病毒（ZIKV）感染后Ku70及miR-124的变化与神经元分化关联。结果 研究发现,Ku70蛋白具有RNA解旋酶活性,并通过其RNA解旋酶活性影响miRNA加工成熟。Ku70缺失引起许多miRNAs上调,其中包括神经细胞特异的miR-124。在人神经前体细胞（hNPCs）和人神经母细胞瘤细胞（SH-SY5Y）中敲低Ku70可促进 miR-124的成熟,从而导致上述细胞向神经元分化。本文进一步发现,ZIKV感染影响了Ku70及miR-124的表达,导致细胞形态的分化。结论 本研究揭示了Ku70的一种新功能,即Ku70有可能参与miRNA的成熟调控和神经细胞的分化,并且可能是ZIKV病毒致小头症的原因之一。     

Intrinsically Disordered Protein Ntr2 Modulates the Spliceosomal RNA Helicase Brr2

Precursor messenger RNA splicing is mediated by the spliceosome, a large and dynamic molecular machine composed of five small nuclear RNAs and numerous proteins. Many spliceosomal proteins are predicted to be intrinsically disordered or contain large disordered regions, but experimental validation of these predictions is scarce, and the precise functions of these proteins are often unclear. Here, we show via circular dichroism spectroscopy, dynamic light scattering, and NMR spectroscopy that the yeast spliceosomal disassembly factor Ntr2 is largely intrinsically disordered. Peptide SPOT analyses, analytical size-exclusion chromatography, and surface plasmon resonance measurements revealed that Ntr2 uses an N-terminal region to bind the C-terminal helicase unit of the Brr2 RNA helicase, an enzyme involved in spliceosome activation and implicated in splicing catalysis and spliceosome disassembly. NMR analyses suggested that Ntr2 does not adopt a tertiary structure and likely remains disordered upon complex formation. RNA binding and unwinding studies showed that Ntr2 downregulates Brr2 helicase activity in vitro by modulating the fraction of helicase molecules productively bound to the RNA substrate. Our data clarify the nature of a physical link between Brr2 and Ntr2, and point to the possibility of a functional Ntr2-Brr2 interplay during splicing.     

SRA的RNA和蛋白质均有转录辅激活功能

类固醇受体激活物（steroid receptor activator, SRA）是一种 类固醇受体辅激活物.最初的研究认为,SRA只存在RNA形式,不存在蛋白质 形式.但是后来的研究发现,SRA是在RNA和蛋白质两个水平上发挥功能的分 子,其cDNA序列存在687 bp保守的核心区域,该核心区域对其发挥转录共激 活活性是必需的.SRA的RNA形式主要参与核受体的转录共激活作用,其表达 与乳腺癌的发生有很大关系,SRA的蛋白质形式（steroid receptor activator protein ,SRAP）也具有类似的功能.但是不同于RNA形式, SRAP可结合到特定基因的启动子区域,并起到阻遏物的作用.本文对SRA的 特点、表达及功能等方面的最新研究进展及其可能的作用机制与作用形式 进行概述.     

Human SCARB2 Transgenic Mice as an Infectious Animal Model for Enterovirus 71

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.     

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

Background

Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed.

Methodology/Principal Findings

In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ² =  44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive.

Conclusions/Significance

The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.     

The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.     

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.     

Human PIEZO1 Ion Channel Functions as a Split Protein

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.     

Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35, 71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (∼10%) mortality rate (69). The ∼30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5′ capped and 3′ polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5′ and 3′ untranslated regions (UTRs). These include two in the 5′ UTR required for BCoV defective interfering (DI) RNA replication (Fig. ​(Fig.1A)1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI region in BCoV (15) has been reanalyzed along with the homologous region in MHV and is now described as comprising SL1 and SL2 (Fig. ​(Fig.1A),1A), of which SL2 has been shown to be a cis-replication structure in the context of the MHV genome (38). In the 3′ UTR, two higher-order cis-replication structures have been identified that function in both DI RNA and the MHV genome. These are a 5′-proximal bulged SL and adjacent pseudoknot that potentially act together as a unit (23, 27, 28, 72) and a 3′-proximal octamer-associated bulged SL (39, 76) (Fig. ​(Fig.1A).1A). In addition, the 5′-terminal 65-nucleotide (nt) leader and the 3′-terminal poly(A) tail have been shown to be cis-replication signals for BCoV DI RNA (15, 60).Open in a separate windowFIG. 1.RNA structures in the BCoV genome tested for nsp1 binding. (A) BCoV 5′-terminal and 3′-terminal cis-acting RNA SL structures and flanking sequences identified for BCoV DI RNA replication. Regions of the genome are identified and SL cis-replication elements are identified schematically. Open boxes at nt 100 and 211 identify AUG start codons for the short upstream ORF and ORF 1, respectively. A closed box at nt 124 identifies the UAG stop codon for the short upstream ORF. Shown below the SL structures are the RNA segments used as ³²P-labeled probes in the gel shift assays. BSL-PK, bulged SL-pseudoknot; 8mer-BSL, octamer-associated bulged SL. (B) Gel shift assays for probes when used with purified nsp1. Protein-RNA complexes identifying a shifted probe are labeled C.In CoVs, the 5′-proximal open reading frame (ORF) of ∼20 kb (called ORF 1) comprising the 5′ two-thirds of the genome is translated to overlapping polyproteins of ∼500 and ∼700 kDa, named pp1a and pp1ab (41). pp1ab is formed by a −1 ribosomal frameshift event at the ORF1a-ORF1b junction during translation (41). pp1a and pp1ab are proteolytically processed into potentially 16 nonstructural protein (nsp) end products or partial end products that are proposed to function together as the replicase (24). ORF 1a encodes nsps 1 to 11 which include papain-like proteases (nsp3), a 3C-like main protease (nsp5), membrane-anchoring proteins (nsps 4 and 6), a potential primase (nsp8), and RNA-binding proteins (nsp 7/nsp 8 complex and nsps 9 and 10) of imprecisely understood function (19, 20, 24, 25, 29, 43, 49, 77). ORF 1b encodes nsps 12 to 16 which function as an RNA-dependent RNA polymerase, a helicase, an exonuclease, an endonuclease, and a 2′-O-methyltransferase, respectively (6, 17, 24, 44). 3′ Proximal genomic ORFs encoding structural and accessory proteins are translated from a 3′-nested set of subgenomic mRNAs (sgmRNAs) (41).The N-terminal ORF 1a protein, nsp1, in the case of BCoV and MHV is also named p28 to identify the cleaved 28-kDa product (18). The precise role of nsp1 in virus replication has not been determined, but it is known that a sequence encoding an N-proximal nsp1 region in MHV (nt 255 to 369 in the 738-nt coding sequence) cannot be deleted from the genome without loss of productive infection (10). nsp1 also directly binds nsp7 and nsp10 (11) and by confocal microscopy is found associated with the membranous replication complex (10, 66) and virus assembly sites (11). The amino acid sequence of nsp1 is poorly conserved among CoVs, indicating that it may be a protein that interacts with cellular components (1, 58). In the absence of other viral proteins, MHV nsp1 induces general host mRNA degradation (79) and cell cycle arrest (16). The SARS-CoV nsp1 homolog, a 20-kDa protein, has been reported to cause mRNA degradation (30, 45), inhibition of host protein synthesis (30, 45, 70), inhibition of interferon signaling (70, 79), and cytokine dysregulation in lung cells (36).In this study, we examine the RNA-binding properties of BCoV nsp1 with the hypothesis that it is a potential regulator of translation or replication through its binding of SLVI mapping within its coding region. The rationale for this hypothesis stems from five observations. (i) In the BCoV DI RNA, the 5′-terminal one-third (approximately) of the nsp1 cistron and the entire nucleocapsid (N) protein cistron together comprise the single contiguous ORF in the DI RNA, and most of both coding regions appear required for DI RNA replication (15). (ii) The partial nsp1 cistron in the DI RNA must be translated in cis for DI RNA replication in helper virus-infected cells (12, 14). (iii) A similar part of the nsp1 cistron is found in the genome of all characterized naturally occurring group 1 and 2 CoV DI RNAs described to date (7, 8). (iv) A cis-acting SL named SLVI is found within the partial nsp1 cistron in the BCoV DI RNA (12). (v) Translation, which involves a 5′→3′ transit of ribosomes, and negative-strand synthesis, which involves a 3′→5′ transit of the RNA-dependent RNA polymerase, cannot simultaneously occur on the same molecule with a single ORF (4, 31). Thus, to enable genome replication an inhibition of translation at least early in infection for cytoplasmically replicating positive-strand RNA viruses is required (4, 5, 22, 32). Mechanisms of translation inhibition have been described for the Qβ viral genome, wherein the viral replicase autoregulates translation by binding an intracistronic cis-replication element (32), and for the polio virus genome, wherein genome circularization inhibits the early translation step (5, 22). Therefore, since nsp1 is synthesized early and also contains an intracistronic cis-replication element, we postulated that it is autoregulatory with RNA binding properties.Here, we do the following: (i) demonstrate by mutagenesis analysis that the 72-nt SLV, mapping immediately upstream of SLVI and within the partial nsp1 cistron, is also a cis-acting DI RNA replication element; (ii) show by gel shift and UV cross-linking analyses that there is likely no binding of an intracellular viral protein to SLV and SLVI (SLV-VI), but there is binding of unidentified cellular proteins of ∼60 and 100 kDa; and (iii) show by gel shift analysis that recombinant nsp1 purified from Escherichia coli does not bind SLV-VI but does bind SLs I to IV in the 5′ UTR and also the 3′-terminal bulged SL in the 3′ UTR, suggesting a possible regulatory role at these sites. Notably, specific binding with ∼2.5 μM affinity of nsp1 to SLIII and its flanking regions in the 5′ UTR was observed. Additionally, we show that, under conditions that would express nsp1 from a DI RNA-encoded sgmRNA, DI RNA levels are greatly reduced; viral RNA species levels, however, are reduced only slightly, and this reduction is transient. These results together indicate that nsp1 is an RNA-binding protein that may function as a regulator of viral translation or replication but not through its binding of cis-acting SLs V and VI within its own cistron.