We described previously that the newly identified HCoV-NL63 virus has a typical coronavirus genome structure and gene order [6]. The nucleotide composition of the genomic (+) strand RNA of several coronaviridae members is presented in Figure 1, demonstrating a common pattern with U as the most abundant nucleotide and G and in particular C as underrepresented nucleotides. HCoV-NL63 has the most extreme nucleotide bias among the coronaviridae, with 39% U and only 14% C. As a general trend, U and C seem to compete directly, because the genomes with the lowest C-count (HCoV-NL63, HCoV-OC43 and BCoV) have the highest U-count and vice versa (Figure 1).

To investigate if all coding regions of HCoV-NL63 display a similarly strong preference for U and against C, we also plotted the nucleotide count for the individual genes and 5' and 3' non-coding regions (Figure 2). The typical nucleotide bias is observed in all genome segments. The highest U-count is found in the ORF3 and E genes (43%) and the lowest C-count in the 1a/1b genes and the 3' UTR (13%, 14% and 14%, respectively). The N gene is most moderate in its nucleotide bias, with 21% C and 32% U, confirming the "competition" idea that was already suggested by inspection of Figure 1.

We plotted the nucleotide distribution along the genome (Figure 3) to determine whether there is any significant variation. We observed that local changes in A-count are inversely linked to changes in G-count. This is most striking in the 20400–26000 nt region, where three A peaks are mirrored by three G anti-peaks. Although the typical bias is maintained along the genome, the most notable variation occurs in the last one-third of the genome, where an increase in C and a decrease in G content is apparent. This region encodes the structural proteins.

Recently, Grigoriev reported an interesting feature within coronaviral genomes that is visible when the cumulative GC-skew is plotted [20,21]. Cumulative GC skew graph is a way to visualize the local G:C ratio along the genome, discarding the local fluctuations. A biphasic pattern was described that separates the 1a/1b polyprotein genes and the structural genes. The cumulative GC-skews for HCoV-NL63 and four other coronaviruses: HCoV-OC43, HCoV-229E, PEDV and SARS-CoV are presented in Figure 4. In the 1a/1b genes, the G:C ratio reaches high levels, whereas for all coronaviruses, including HCoV-NL63, the 3' end of the genome displays a flattening of the curve, as the G:C ratio reaches value ~ 1 or less. Grigoriev proposed that this biphasic pattern is due to the discontinuous transcription process [20]. He suggested that the frequent deamination of cytosine on the (-) strand RNA results in a decrease of G on the (+) strand in the region encoding the structural genes. In the discussion section we will present an alternative mechanistic explanation.

The bias in the nucleotide count led us to compare the codon usage of HCoV-NL63 with that of human mRNA (Table 1). The codon usage of HCoV-NL63 differs markedly from that of human mRNAs. Third-base choices in the four-codon families (Thr, Pro, Ala, Gly, Val) provide a convenient example of this contrasting codon usage. For instance, the Gly codons in human mRNAs prefer C (34%) over G (25%), A (25%) and U (16%). In contrast, HCoV-NL63 prefers U (83%) over A (7%), C (8%) and G (2%). This result strongly suggests that the codon usage is shaped directly by the unusual nucleotide composition of the viral genome, that is a high U-count and a low G/C-count. All HCoV-NL63 genes, except for the E gene, follow this trend (Table 1). The coronaviral addiction to the U nucleotide is most prominent in the "free" third position of degenerate codons. For the complete genome, the U-count at the third position is up to 58% whereas the A-count is 20%, G-count is 13% and C-count is only 9% (Figure 5). This illustrates that the U-pressure mainly affects the %C and %G.

The 5' end of HCoV-NL63 genome RNA contains the L sequence of 72 nucleotides that ends with the L TRS element. This TRS has a high similarity to short sequences that are located in front of each open reading frame (S-ORF3-E-M-N) [22]. We previously identified the L TRS and body TRS of the N gene using a cDNA bank [6], which allowed us to predict the body TRS of the other genes. To confirm these predictions, we amplified and sequenced all sg mRNA fragments with a general L primer and gene-specific 3' primers in an RT-PCR protocol.

Inspection of sg mRNA junctions indicated that they are indeed composed of the part of the HCoV-NL63 genome that is directly downstream of a particular body TRS, with its 5' end derived from the leader sequence. Apparently, strand transfer occurred on the 5' end of the body TRS, as indicated in Figure 6. The most conserved TRS region was defined by multiple sequence alignment as AACUAAA (gray box). This core sequence is conserved in all sg mRNA, except for the E gene that contains the sub-optimal TRS core AACUAUA (Figure 6). Interestingly, the E gene contains a 13-nucleotide sequence upstream of the core sequence with perfect homology to the L sequence. Perhaps the upstream sequence compensates for the absence of an optimal TRS core during discontinuous (-) strand synthesis. This would suggest that these sequences are copied during (-) strand synthesis, and that the actual strand transfer within the E sequences occurred after copying of the core TRS and the next 13 nucleotides. Evidence for such a "delayed" strand transfer is provided by the junction analysis of the M and N sg mRNAs, which clearly demonstrates that the nucleotides directly upstream of the core TRS are derived from the body TRS element and not from the leader.

To determine whether the predicted sg mRNAs encoding the S-ORF3-E-M-N proteins are produced in virus-infected cells, we performed Northern blot analysis on total cellular RNA (Figure 7). We used a (-) strand N gene probe that anneals to both genomic RNA and all sg (+) strand mRNAs. We included RNA from MHV-infected cells to obtain discrete size markers. Six distinct mRNAs are produced in HCoV-NL63 infected cells. The sizes of the RNA fragments were estimated and these values nicely fit the size of the genomic RNA and the five predicted sg mRNAs. All HCoV-NL63 ORFs that have the potential to encode viral proteins are indeed transcribed into sg mRNAs (Figure 7).

To determine the expression level of each subgenomic RNA, we measured the intensity of the signals. When plotted as a function of the genome position (Figure 8), there appears a correlation between the relative distance of a gene to the 3' terminus and its RNA expression level, with the exception of the E gene.

The nucleotide content of different Coronaviridae family members was assessed using BioEdit software. The nucleotide distribution was determined using a Microsoft Excel datasheet (300 nucleotide (nt) window and 10-nt step). Codon usage was assessed using DNA 2.0 software. Data was processed in Microsoft Excel datasheet and all statistical analysis was performed with SPSS 11.5.0 software. The level of significance of the nucleotide bias was established for 300-nt non-overlapping windows with the non-parametric Mann-Whitney U test for two independent samples. Cumulative GC-skew graphs were generated as described previously [20] with the value in step n defined as the sum of (G-C)/(G+C) from step 0 to n (200-nt sliding window, 10-nt step).

HCoV-NL63 RNA was obtained from virus-infected LLC-MK2 cells (2 × 10⁷) after 6 days of culture (virus passage 7). Mouse Hepatitis Virus (MHV) RNA was obtained by infecting 2 × 10⁷ LR7 cells with MHV strain A59. The medium was removed and the cells were dissolved in 15 ml TRIzol^® and RNA was isolated according to the standard TRIzol^® procedure. RNA was subsequently precipitated with 0.8 volume of isopropanol, dried and dissolved in 50 μl H₂O. Integrity of the RNA was analyzed by electrophoresis on a non-denaturating 0.8% agarose gel. RNA was stored at -150°C.

The cDNA used for sequencing and probe construction was made by MMLV-RT on viral RNA with 1 μg of random hexamer DNA primers in 10 mM Tris pH 8.3, 50 mM KCl, 0.1% Triton-X100, 6 mM of MgCl₂ and 50 μM of each dNTPs at 37°C for 1 hour. The single stranded cDNA product was made into double-stranded DNA in a standard PCR reaction with 1.25 U of Taq polymerase (Perkin-Elmer) per reaction with appropriate primers (see below).

Gel electrophoresis of viral RNA was performed on a 1% agarose gel with 7% of formaldehyde at 100 Volt in 1×MOPS buffer (40 mM MOPS, 10 mM sodium acetate, pH 7.0). Transfer onto a positively charged nylon membrane (Boehringer Mannheim) was done overnight by means of capillary force. RNA was linked to the membrane in a UV crosslinker (Stratagene). For generation of the HCoV-NL63 probe, the RT-PCR product was further amplified with 5' primer N5PCR1 (CTG TTA CTT TGG CTT TAA AGA ACT TAG G) and 3' primer N3PCR1 (CTC ACT ATC AAA GAA TAA CGC AGC CTG). Similarly, the MHV probe was amplified with 5' primer MHV_UTR-B5' (GAT GAA GTA GAT AAT GTA AGC GT) and 3' primer MHV_UTR-B3' (TGC CAC AAC CTT CTC TAT CTG TTA T). Labeling of the probes was done in a standard PCR reaction with specific 3' primers (N3PCR1 and MHV_UTR-B3') in presence of [α-³²P]dCTP. Prehybridization and hybridization was done in ULTRAhyb buffer (Ambion) at 50°C for 1 and 12 hours, respectively. The membrane was then washed at room temperature with low-stringency buffer (2×SSC, 0.2% SDS) and at 50°C in high stringency buffer (0.1×SSC, 0.2% SDS). Images were obtained using the STORM 860 phosphorimager (Amersham Biosciences) and data analysis was performed with the ImageQuant software package. The size of sg mRNA fragments of HCoV-NL63 were estimated from their migration on the Northern blot using the sg mRNA of MHV as size marker.

The L/body TRS junctions were PCR-amplified from an HCoV-NL63 cDNA bank. We performed 35 cycle PCR with the 5' L primer (L5 – TAA AGA ATT TTT CTA TCT ATA GAT AG) and gene specific 3' primers (S gene – SL3' – ACT ACG GTG ATT ACC AAC ATC AAT ATA; ORF3 – 4L3' – CAA GCA ACA CGA CCT CTA GCA GTA AG; E gene – EL3' – TAT TTG CAT ATA ATC TTG GTA AGC; M gene – ML3' – GAC CCA GTC CAC ATT AAA ATT GAC A; N gene – 3-163-F15 – ATT ACC TAG GTA CTG GAC CT). The PCR products were analyzed by electrophoresis on a 0.8% agarose gel and products of discrete size were used for sequencing using the BigDye terminator kit (ABI) and ABI Prism 377 sequencer (Perkin Elmer). Sequence analysis was performed by Sequence Navigator and AutoAssembler 2.1 software.

The complete genome sequence of HCoV-NL63 [6] is deposited in GenBank (accession number: NC_005831). sg mRNA sequences are deposited in GenBank under the accession numbers: AY697419-AY697423. The GenBank accession number of the sequences used in this genome analysis are: MHV (mouse hepatitis virus, strain MHV-A59): NC_001846; HCoV-229E: NC_002645; HCoV-OC43 strain ATCC VR-759: NC_005147; PEDV (porcine epidemic diarrhea virus, strain CV777): AF353511; TGEV (transmissible gastroenteritis virus, strain Purdue): NC_002306; SARS-CoV isolate Tor2: NC_004718; IBV (avian infectious bronchitis virus, strain Beaudette): NC_001451; BCoV (bovine coronavirus, isolate BCoV-ENT): NC_003045.