RNA recognition and cleavage by the SARS coronavirus endoribonuclease

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.     

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.     

Object-oriented biological system integration: a SARS coronavirus example

MOTIVATION: The importance of studying biology at the system level has been well recognized, yet there is no well-defined process or consistent methodology to integrate and represent biological information at this level. To overcome this hurdle, a blending of disciplines such as computer science and biology is necessary. RESULTS: By applying an adapted, sequential software engineering process, a complex biological system (severe acquired respiratory syndrome-coronavirus viral infection) has been reverse-engineered and represented as an object-oriented software system. The scalability of this object-oriented software engineering approach indicates that we can apply this technology for the integration of large complex biological systems. AVAILABILITY: A navigable web-based version of the system is freely available at http://people.musc.edu/~zhengw/SARS/Software-Process.htm     

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.     

Prediction of functional class of the SARS coronavirus proteins by a statistical learning method

The complete genome of severe acute respiratory syndrome coronavirus (SARS-CoV) reveals the existence of putative proteins unique to SARS-CoV. Identification of their function facilitates a mechanistic understanding of SARS infection and drug development for its treatment. The sequence of the majority of these putative proteins has no significant similarity to those of known proteins, which complicates the task of using sequence analysis tools to probe their function. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to SARS-CoV proteins. Testing results indicate that SVM is able to predict the functional class of 73% of the known SARS-CoV proteins with available sequences and 67% of 18 other novel viral proteins. A combination of the sequence comparison method BLAST and SVMProt can further improve the prediction accuracy of SMVProt such that the functional class of two additional SARS-CoV proteins is correctly predicted. Our study suggests that the SARS-CoV genome possibly contains a putative voltage-gated ion channel, structural proteins, a carbon-oxygen lyase, oxidoreductases acting on the CH-OH group of donors, and an ATP-binding cassette transporter. A web version of our software, SVMProt, is accessible at http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi .     

Development of the magnetic capture system for recovery of Xiphinema americanum

Xiphinema americanum is listed as a quarantine nematode by The European Plant Protection Organisation because of its ability to transmit quarantine viruses. Detection of this nematode in soil samples, or in mixed nematode samples extracted from soil, is therefore of paramount importance. We recently described the use of magnetic beads (Dynabeads) in combination with probes such as lectins or antibodies for recovery of small endoparasitic nematodes including Meloidogyne spp. and Globodera rostochiensis from mixed nematode samples. Here we show that magnetic capture can be used with much larger nematodes such as X. americanum. We describe lectins and antisera that bind to the surface of this nematode and show that the antisera can be used in the Dynabeads system to recover and enrich X. americanum from mixed nematode samples.     

Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million‐fold improvements in binding affinity by alternating rounds of diversification and flow cytometry‐based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads—allowing isolation of extremely weak binders from billions of non‐binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000‐fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin‐biotin that do not cross‐react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Influenza as a model system for studying the cross-species transfer and evolution of the SARS coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) moved into humans from a reservoir species and subsequently caused an epidemic in its new host. We know little about the processes that allowed the cross-species transfer of this previously unknown virus. I discuss what we have learned about the movement of viruses into humans from studies of influenza A, both how it crossed from birds to humans and how it subsequently evolved within the human population. Starting with a brief review of severe acute respiratory syndrome to highlight the kinds of problems we face in learning about this viral disease, I then turn to influenza A, focusing on three topics. First, I present a reanalysis of data used to test the hypothesis that swine served as a "mixing vessel" or intermediate host in the transmission of avian influenza to humans during the 1918 "Spanish flu" pandemic. Second, I review studies of archived viruses from the three recent influenza pandemics. Third, I discuss current limitations in using molecular data to study the evolution of infectious disease. Although influenza A and SARS-CoV differ in many ways, our knowledge of influenza A may provide important clues about what limits or favours cross-species transfers and subsequent epidemics of newly emerging pathogens.     

Subtractive hybridization magnetic bead capture: a new technique for the recovery of full-length ORFs from the metagenome

A new method for the recovery of full-length open reading frames from metagenomic nucleic acid samples is reported. This technique, based on subtractive hybridization magnetic bead capture technology, has the potential to access multiple gene variants from a single amplification reaction. It is now widely accepted that classical microbiological methods provide only limited access to the true microbial biodiversity (less than 1%). The desire to access a higher proportion of the metagenome has led to the development of efficient environmental nucleic acid extraction technologies and to a range of sequence-dependent and sequence-independent gene discovery techniques. These methods avoid many of the limitations of culture-dependent gene targeting.     

Biochemical characterization of exoribonuclease encoded by SARS coronavirus

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires Mg2+ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.     

斑马鱼总RNA提取和纯化

采取异硫氰酸胍法从斑马鱼的3个样品中提取和纯化RNA,结果表明,RNAA260分别为0、587、0．719、0．151.A260/A280比值分别为1．89、1．92、1．82。电泳条带也清晰可见,可以用于进一步的分子生物学研究。     

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.     

Immunopathogenesis of coronavirus infections: implications for SARS

At the end of 2002, the first cases of severe acute respiratory syndrome (SARS) were reported, and in the following year, SARS resulted in considerable mortality and morbidity worldwide. SARS is caused by a novel species of coronavirus (SARS-CoV) and is the most severe coronavirus-mediated human disease that has been described so far. On the basis of similarities with other coronavirus infections, SARS might, in part, be immune mediated. As discussed in this Review, studies of animals that are infected with other coronaviruses indicate that excessive and sometimes dysregulated responses by macrophages and other pro-inflammatory cells might be particularly important in the pathogenesis of disease that is caused by infection with these viruses. It is hoped that lessons from such studies will help us to understand more about the pathogenesis of SARS in humans and to prevent or control outbreaks of SARS in the future.     

Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies

Background

Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).

Methodology/Principal Findings

We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.

Conclusions/Significance

The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.     

Expression cloning of functional receptor used by SARS coronavirus

We have expressed a series of truncated spike (S) glycoproteins of SARS-CoV and found that the N-terminus 14-502 residuals were sufficient to bind to SARS-CoV susceptible Vero E6 cells. With this soluble S protein fragment as an affinity ligand, we screened HeLa cells transduced with retroviral cDNA library from Vero E6 cells and obtained a HeLa cell clone which could bind with the S protein. This cell clone was susceptible to HIV/SARS pseudovirus infection and the presence of a functional receptor for S protein in this cell clone was confirmed by the cell-cell fusion assay. Further studies showed the susceptibility of this cell was due to the expression of endogenous angiotensin-converting enzyme 2 (ACE2) which was activated by inserted LTR from retroviral vector used for expression cloning. When human ACE2 cDNA was transduced into NIH3T3 cells, the ACE2 expressing NIH3T3 cells could be infected with HIV/SARS pseudovirus. These data clearly demonstrated that ACE2 was the functional receptor for SARS-CoV.     

An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus

The −1 ribosomal frameshifting requires the existence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA. An atypical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H-type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus (SARS CoV) genome. This pseudoknot can serve as an efficient stimulator for −1 frameshifting in vitro. Mutational analysis of the extra stem suggests frameshift efficiency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus-type pseudoknot. More importantly, an upstream RNA sequence separated by a linker 5′ to the slippery site is also identified to be capable of modulating the −1 frameshift efficiency. RNA sequence containing this attenuation element can downregulate −1 frameshifting promoted by an atypical pseudoknot of SARS CoV and two other pseudoknot stimulators. Furthermore, frameshift efficiency can be reduced to half in the presence of the attenuation signal in vivo. Therefore, this in cis RNA attenuator represents a novel negative determinant of general importance for the regulation of −1 frameshift efficiency, and is thus a potential antiviral target.     

Potent and selective inhibition of SARS coronavirus replication by aurintricarboxylic acid

The severe acute respiratory syndrome virus (SARS) is a coronavirus that instigated regional epidemics in Canada and several Asian countries in 2003. The newly identified SARS coronavirus (SARS-CoV) can be transmitted among humans and cause severe or even fatal illnesses. As preventive vaccine development takes years to complete and adverse reactions have been reported to some veterinary coronaviral vaccines, anti-viral compounds must be relentlessly pursued. In this study, we analyzed the effect of aurintricarboxylic acid (ATA) on SARS-CoV replication in cell culture, and found that ATA could drastically inhibit SARS-CoV replication, with viral production being 1000-fold less than that in the untreated control. Importantly, when compared with IFNs alpha and beta, viral production was inhibited by more than 1000-fold as compared with the untreated control. In addition, when compared with IFNs alpha and beta, ATA was approximately 10 times more potent than IFN alpha and 100 times more than interferon beta at their highest concentrations reported in the literature previously. Our data indicated that ATA should be considered as a candidate anti-SARS compound for future clinical evaluation.     

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.