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. 2004 Nov 17:1:7.
doi: 10.1186/1743-422X-1-7.

Genome structure and transcriptional regulation of human coronavirus NL63

Krzysztof Pyrc  1 , Maarten F Jebbink, Ben Berkhout, Lia van der Hoek
Affiliations

Genome structure and transcriptional regulation of human coronavirus NL63

Krzysztof Pyrc et al. Virol J. .

Abstract

Background: Two human coronaviruses are known since the 1960s: HCoV-229E and HCoV-OC43. SARS-CoV was discovered in the early spring of 2003, followed by the identification of HCoV-NL63, the fourth member of the coronaviridae family that infects humans. In this study, we describe the genome structure and the transcription strategy of HCoV-NL63 by experimental analysis of the viral subgenomic mRNAs.

Results: The genome of HCoV-NL63 has the following gene order: 1a-1b-S-ORF3-E-M-N. The GC content of the HCoV-NL63 genome is extremely low (34%) compared to other coronaviruses, and we therefore performed additional analysis of the nucleotide composition. Overall, the RNA genome is very low in C and high in U, and this is also reflected in the codon usage. Inspection of the nucleotide composition along the genome indicates that the C-count increases significantly in the last one-third of the genome at the expense of U and G. We document the production of subgenomic (sg) mRNAs coding for the S, ORF3, E, M and N proteins. We did not detect any additional sg mRNA. Furthermore, we sequenced the 5' end of all sg mRNAs, confirming the presence of an identical leader sequence in each sg mRNA. Northern blot analysis indicated that the expression level among the sg mRNAs differs significantly, with the sg mRNA encoding nucleocapsid (N) being the most abundant.

Conclusions: The presented data give insight into the viral evolution and mutational patterns in coronaviral genome. Furthermore our data show that HCoV-NL63 employs the discontinuous replication strategy with generation of subgenomic mRNAs during the (-) strand synthesis. Because HCoV-NL63 has a low pathogenicity and is able to grow easily in cell culture, this virus can be a powerful tool to study SARS coronavirus pathogenesis.

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Figures

Figure 1
Figure 1
Nucleotide content of coronaviridae RNA genomes. We arranged the viruses based on their C-count, which ranges from 14% (HCoV-NL63) to 20% (SARS-CoV).
Figure 2
Figure 2
Nucleotide content of individual HCoV-NL63 genes and the 5'/3' untranslated regions (UTR).
Figure 3
Figure 3
Nucleotide distribution along the HCoV-NL63 genome. The change in the C- and G-count at two-third of the genome is statistically significant for all tested coronaviruses (HCoV-NL63, HCoV-229E, SARS-CoV, HCoV-OC43) with p < 0.01 for C-count and p < 0.05 for G-count in Mann-Whitney U test for two independent samples.
Figure 4
Figure 4
Cumulative GC-skew diagrams for several coronaviral RNA genomes. The vertical bar indicates the border between the 1a/1b and the structural genes.
Figure 5
Figure 5
Nucleotide composition of the first, second and third codon positions in the HCoV-NL63 genome.
Figure 6
Figure 6
Body-leader junctions of all HCoV-NL63 sg mRNAs. Shown on top is the leader (L) sequence and below the specific sequences upstream of the structural genes. The fusion of 5' L sequences to 3' sg RNA is indicated by the boxes. Sequence homology between the strands near the junction is marked by asterisks, the conserved AACUAAA TRS core is highlighted in gray.
Figure 7
Figure 7
The left panel shows the Northern blot analysis of HCoV-NL63 RNA in infected LLC-MK2 cells. RNA of HCoV-NL63 (NL63 lane) was compared with RNA of MHV strain A59 (MHV lane). Non-infected LLC-MK2 cells are included as a negative control (control lane). MHV RNA bands represent the complete genome (1) and sg mRNAs 2a (2), S (3), 17.8 (4), 13.1 and E (5), M (6), N (7). HCoV-NL63 RNA includes the complete genome (1) and sg mRNAs for S (2), ORF3 (3), E (4), M (5) and N (6). The right panel shows the MHV and HCoV-NL63 genome organization and the HCoV-NL63 sg-mRNAs.
Figure 8
Figure 8
Expression levels of the HCoV-NL63 genomic and sg mRNAs.
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