Bioinformatics analysis of SARS coronavirus genome polymorphism

Background  

We have compared 38 isolates of the SARS-CoV complete genome. The main goal was twofold: first, to analyze and compare nucleotide sequences and to identify positions of single nucleotide polymorphism (SNP), insertions and deletions, and second, to group them according to sequence similarity, eventually pointing to phylogeny of SARS-CoV isolates. The comparison is based on genome polymorphism such as insertions or deletions and the number and positions of SNPs.     

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.     

Moderate mutation rate in the SARS coronavirus genome and its implications

Background

The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) and multiple genomic sequences have been revealed since mid-April, 2003. After a quiet summer and fall in 2003, the newly emerged SARS cases in Asia, particularly the latest cases in China, are reinforcing a wide-spread belief that the SARS epidemic would strike back. With the understanding that SARS-CoV might be with humans for years to come, knowledge of the evolutionary mechanism of the SARS-CoV, including its mutation rate and emergence time, is fundamental to battle this deadly pathogen. To date, the speed at which the deadly virus evolved in nature and the elapsed time before it was transmitted to humans remains poorly understood.

Results

Sixteen complete genomic sequences with available clinical histories during the SARS outbreak were analyzed. After careful examination of multiple-sequence alignment, 114 single nucleotide variations were identified. To minimize the effects of sequencing errors and additional mutations during the cell culture, three strategies were applied to estimate the mutation rate by 1) using the closely related sequences as background controls; 2) adjusting the divergence time for cell culture; or 3) using the common variants only. The mutation rate in the SARS-CoV genome was estimated to be 0.80 – 2.38 × 10^-3 nucleotide substitution per site per year which is in the same order of magnitude as other RNA viruses. The non-synonymous and synonymous substitution rates were estimated to be 1.16 – 3.30 × 10^-3 and 1.67 – 4.67 × 10^-3 per site per year, respectively. The most recent common ancestor of the 16 sequences was inferred to be present as early as the spring of 2002.

Conclusions

The estimated mutation rates in the SARS-CoV using multiple strategies were not unusual among coronaviruses and moderate compared to those in other RNA viruses. All estimates of mutation rates led to the inference that the SARS-CoV could have been with humans in the spring of 2002 without causing a severe epidemic.

     

Assessment of putative protein targets derived from the SARS genome

The ability to rapidly and reliably develop hypotheses on the function of newly discovered protein sequences requires systematic and comprehensive analysis. Such an analysis, embodied within the DS GeneAtlas pipeline, has been used to critically evaluate the severe acute respiratory syndrome (SARS) genome with the goal of identifying new potential targets for viral therapeutic intervention. This paper discusses several new functional hypotheses on the roles played by the constituent gene products of SARS, and will serve as an example of how such assignments can be developed or extended on other systems of interest.     

Sequence specificity of mRNA N6-adenosine methyltransferase

The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells.     

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (A_m), generating N6, 2′O-dimethyladenosine (m⁶A_m) when A_m is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and A_m), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated A_m over unmodified A nucleosides is due mainly to increased binding affinity for A_m. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-A_m oligonucleotide with approximately the same affinity as that of a cap analog (K_M = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-A_m with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.     

Purification of BsuE methyltransferase and its application in genome mapping.

We have used a combination of BsuE methyltransferase (M-BsuE) and NotI restriction enzyme to cut genomic DNA at a subset of NotI sites. The usefulness of this system is shown in a re-examination of the restriction map of the human MHC. Combinations of methylases and restriction enzymes can be used to generate cuts at different frequencies in genomic DNA, such that they generate ends complementary to NotI ends, and can be used in conjunction with NotI linking clones in chromosome jumping experiments. These enzyme combinations have the potential to produce cutting sites in genomic DNA spaced at intervals favorable for extensive mapping, fragment enrichment, and cloning efforts.     

Mutational patterns correlate with genome organization in SARS and other coronaviruses

Focused efforts by several international laboratories have resulted in the sequencing of the genome of the causative agent of severe acute respiratory syndrome (SARS), novel coronavirus SARS-CoV, in record time. Using cumulative skew diagrams, I found tht mutational patterns in the SARS-CoV genome were strikingly different from other coronaviruses in terms of mutation rates, although they were in general agreement with the model of the coronavirus lifecycle. These findings might be relevant for the development of sequence-based diagnostics and the design of agents to treat SARS.     

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds

Phytochrome‐interacting factor 1 (PIF1) inhibits light‐dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyB_off condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high‐level expression of PIF1 mRNA, the expression pattern of PIF1 target genes, and the light‐dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light‐dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high‐level expression of PIF1 mRNA in imbibed seeds.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system.

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

The structure of a rigorously conserved RNA element within the SARS virus genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.     

Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.     

Yeast-based genetic system for functional analysis of poxvirus mRNA cap methyltransferase

Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Delta cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Delta complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.