High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.

The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

A new cistron in the murine hepatitis virus replicase gene.

We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

Evaluation of real-time RT-PCR for the quantification of FCoV shedding in the faeces of domestic cats.

Faecal samples were taken from cats living in multi-cat households with endemic feline coronavirus (FCoV) infection. Total RNA was extracted from faecal suspensions and FCoV RNA was quantified using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The real-time RT-PCR threshold cycle (C(T)) values were consistently high suggesting that the samples contained very little viral RNA. However, experiments in which RNA extracted from FCoV-infected cell culture supernatants was combined with RNA extracted from faecal suspensions revealed the presence of faecal factors that significantly inhibited the reverse transcription reaction. Consequently, three methods of RNA extraction were investigated and RNA dilution was undertaken to investigate whether the effects of the faecal inhibitors could be reduced. Our results show that using the QIAgen RNA mini kit for RNA extraction and dilution of the RNA samples helps to reduce the inhibitory effects. However, because the extent of the inhibitory effects varied between faecal samples, accurate quantification proved difficult. We, therefore, conclude that although real-time RT-PCR provides an excellent method for detecting the presence of viral shedding, quantification of FCoV RNA in faecal material has to take into account the possible effects of RT-PCR inhibitors. It is, therefore, essential that all new assays, and the methods of sample preparation, are carefully evaluated before being used in a clinical setting.

Type I feline coronavirus spike glycoprotein fails to recognize aminopeptidase N as a functional receptor on feline cell lines.

There are two types of feline coronaviruses that can be distinguished by serology and sequence analysis. Type I viruses, which are prevalent in the field but are difficult to isolate and propagate in cell culture, and type II viruses, which are less prevalent but replicate well in cell culture. An important determinant of coronavirus infection, in vivo and in cell culture, is the interaction of the virus surface glycoprotein with a cellular receptor. It is generally accepted that feline aminopeptidase N can act as a receptor for the attachment and entry of type II strains, and it has been proposed that the same molecule acts as a receptor for type I viruses. However, the experimental data are inconclusive. The aim of the studies reported here was to provide evidence for or against the involvement of feline aminopeptidase N as a receptor for type I feline coronaviruses. Our approach was to produce retroviral pseudotypes that bear the type I or type II feline coronavirus surface glycoprotein and to screen a range of feline cell lines for the expression of a functional receptor for attachment and entry. Our results show that type I feline coronavirus surface glycoprotein fails to recognize feline aminopeptidase N as a functional receptor on three continuous feline cell lines. This suggests that feline aminopeptidase N is not a receptor for type I feline coronaviruses. Our results also indicate that it should be possible to use retroviral pseudotypes to identify and characterize the cellular receptor for type I feline coronaviruses.

Genomic RNA sequence of feline coronavirus strain FCoV C1Je.

This paper reports the first genomic RNA sequence of a field strain feline coronavirus (FCoV). Viral RNA was isolated at post mortem from the jejunum and liver of a cat with feline infectious peritonitis (FIP). A consensus sequence of the jejunum-derived genomic RNA (FCoV C1Je) was determined from overlapping cDNA fragments produced by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. RT-PCR products were sequenced by a reiterative sequencing strategy and the genomic RNA termini were determined using a rapid amplification of cDNA ends PCR strategy. The FCoV C1Je genome was found to be 29,255 nucleotides in length, excluding the poly(A) tail. Comparison of the FCoV C1Je genomic RNA sequence with that of the laboratory strain FCoV FIP virus (FIPV) 79-1146 showed that both viruses have a similar genome organisation and predictions made for the open reading frames and cis-acting elements of the FIPV 79-1146 genome hold true for FCoV C1Je. In addition, the sequence of the 3'-proximal third of the liver derived genomic RNA (FCoV C1Li), which encompasses the structural and accessory protein genes of the virus, was also determined. Comparisons of the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences revealed 100% nucleotide identity, a finding that questions the well accepted 'internal mutation theory' of FIPV pathogenicity.

Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication.

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.

Genomic RNA sequence of Feline coronavirus strain FIPV WSU-79/1146.

A consensus sequence of the Feline coronavirus (FCoV) (strain FIPV WSU-79/1146) genome was determined from overlapping cDNA fragments produced by RT-PCR amplification of viral RNA. The genome was found to be 29 125 nt in length, excluding the poly(A) tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to that of other coronaviruses. The genomic RNA was analysed for putative cis-acting elements and the pattern of subgenomic mRNA synthesis was analysed by Northern blotting. Comparative sequence analysis of the predicted FCoV proteins identified 16 replicase proteins (nsp1-nsp16) and four structural proteins (spike, membrane, envelope and nucleocapsid). Two mRNAs encoding putative accessory proteins were also detected. Phylogenetic analyses confirmed that FIPV WSU-79/1146 belongs to the coronavirus subgroup G1-1. These results confirm and extend previous findings from partial sequence analysis of FCoV genomes.

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, M(pro), a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus M(pro) is reported. The structure was refined to 1.96 A resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that M(pro) self-processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin-like fold that is connected by a 16 residue loop to an extra domain featuring a novel alpha-helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of M(pro) substrates. Interactions involving the N-terminus and the alpha-helical domain stabilize the loop in the orientation required for trans-cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.