Linda A. Guarino

The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor

ABSTRACT Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene.

An early gene which augments the expression of the delayed early/late 39K gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was identified by functional mapping. Transient expression of the plasmid p39CAT, containing the bacterial chloramphenicol acetyltransferase coding sequences under the control of the promoter of the 39K protein, was observed in cells cotransfected with AcNPV DNA digested with several restriction endonucleases. However, when p39CAT was cotransfected with viral DNA digested with Bg/II restriction endonuclease, no CAT activity could be detected. To map the location of the Bg/II-sensitive sequences required for efficient expression of 39CAT, p39CAT and Bg/II-digested viral DNA were cotransfected with a PstI library of AcNPV DNA. The PstI-N fragment restored 39CAT activity. A major early 1.3-kb transcript from this fragment was mapped by S1 nuclease analysis. Transient assay experiments indicated that this major transcript of the PstI-N fragment was produced by an immediate early gene, named IE-N. The PstI-N fragment alone did not activate expression of p39CAT but was required when IE-1 was present in limiting quantities.

Molecular analysis of a baculovirus regulatory gene.

To better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of IE-N expressed by Autographa californica nuclear polyhedrosis virus was determined. The 2.0-kb PstI-EcoRV restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early IE-N gene. Using a convenient restriction site, the 285-bp promoter of IE-N was divided into two functional regions as defined by transient expression assays of mutant sequences. The sequences of IE-N from -1 to -45 nt encoded a minimal promoter capable of directing low levels of transcription. The minimal promoter was fully responsive to positive regulation by IE-N. The upstream region from -46 to -285 nt contains two direct repeats which increased levels of IE-N gene expression. Computer-assisted translation of the IE-N sequence indicates that this fragment of DNA encodes a single long open reading frame with a predicted molecular weight of 47,000. The amino acid sequence of the predicted protein exhibits three motifs common to transcriptional regulators: a serine-threonine rich region, a proline-rich region, and a polyglutamine tract. IE-N autoregulates its own expression and stimulates both IE-1 and IE-0 in transient assays. The stimulation of IE-1 may account for the augmenting activity of IE-N in the IE-1-mediated trans-activation of the 39K promoter.

Structure of an endoglucanase from termite, Nasutitermes takasagoensis.

Contrary to conventional wisdom, it has been shown recently that termites do not necessarily depend on symbiotic bacteria to process cellulose. They secrete their own cellulases, mainly endo-beta-1,4-glucanase and beta-1,4-glucosidase. Here, the first structure of an endogenous endoglucanase from the higher termite Nasutitermes takasagoensis (NtEgl) is reported at 1.40 A resolution. NtEgl has the general folding of an (alpha/alpha)(6) barrel, which is a common folding pattern for glycosyl hydrolase family 9. Three-dimensional structural analysis shows that the conserved Glu412 is the catalytic acid/base residue and the conserved Asp54 or Asp57 is the base. The enzyme has a Ca(2+)-binding site near its substrate-binding cleft. Comparison between the structure of the Ca(2+)-free enzyme produced by reducing the pH of the soaked crystal from 5.6 (the pH of optimum enzyme activity) to 2.5 with that of the Ca(2+)-bound enzyme did not show significant differences in the locations of the C(alpha) atoms. The main differences are in the conformation of the residue side chains ligating the Ca(2+) ion. The overall structure of NtEgl at pH 6.5 is similar to that at pH 5.6. The major change observed was in the conformation of the side chain of the catalytic acid/base Glu412, which rotates from a hydrophobic cavity to a relatively hydrophilic environment. This side-chain displacement may decrease the enzyme activity at higher pH.

Mutational Analysis of the SARS Virus Nsp15 Endoribonuclease: Identification of Residues Affecting Hexamer Formation

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.

Nucleotide sequence and characterization of the 39K gene region of Autographa californica nuclear polyhedrosis virus.

The complete nucleotide sequence of the Pstl-K fragment of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. This region of the genome contains the delayed early 39K gene and V-ubi, a viral protein with homology to ubiquitine. In addition, Pstl-K potentially encodes five other proteins. Hybrid-select translation mapped nine viral-specific proteins to this fragment. Three proteins were observed with both early and late RNA; two of them comigrated with proteins directed by transcripts made from the 39K open reading frame (ORF). One protein which was only seen with hybrid-selected late RNA comigrated with ubiquitin. A correction of previously published sequence data indicates that there is an additional AUG codon upstream of the 39K ORF. This AUG codon is located 8 nucleotides downstream of the early transcription initiation site. The methionine codon is followed by codons for three amino acids and a termination codon. The 39K ORF is located 102 nucleotides downstream of the minicistron. In vitro transcription-translation experiments confirmed that the downstream AUG serves as the initiation codon for the 39K ORF. Radioimmunoprecipitation experiments indicated that the Pstl-K-encoded 39K protein reacted with antiserum raised against a previously described nuclear matrix-associated 39K protein.

Biochemical and Genetic Analyses of Murine Hepatitis Virus Nsp15 Endoribonuclease

ABSTRACT The goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (MHV) Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT virus) on several different cell lines. Interestingly, viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT virus. Cells infected with mNsp15 mutant viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.

The Autographa californica Nuclear Polyhedrosis Virus p143 Gene Encodes a DNA Helicase

ABSTRACT The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had aKm of 60 μM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.

Identification of spliced baculovirus RNAs expressed late in infection

Previous to this study, the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was known to express only one spliced RNA (spliced IE1 or IE0). We have conducted an analysis of RNA expressed during infection of Spodoptera frugiperda cells with AcMNPV and have identified a set of five additional spliced RNAs expressed late in infection. A reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the LS (late, spliced) RNAs. S1 nuclease and primer extension analyses were used to map the transcription initiation sites of LS RNAs. LS1 and LS2 initiated at positions -138 and -117, respectively (relative to the IE0 +1 transcription start site). Both LS1 and LS2 contain an additional cistron potentially encoding a small, highly basic polypeptide. LS3 (-79), LS4 (-22), and LS5 (+51/52) RNAs encode only the predicted downstream IE0 ORF. Although several baculovirus late gene consensus transcription initiation sites (ATAAG) were identified within this region, only LS5 initiated at one of these conserved motifs. An S1 nuclease analysis was done to determine whether unspliced precursors of LS RNAs could be detected. Early in infection, a greater proportion of IE0 RNA was detected in the spliced form; however, during the late phase of infection a significantly greater amount of unspliced precursor forms of LS RNAs was observed.

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15.

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.