Elements in the canine distemper virus M 3' UTR contribute to control of replication efficiency and virulence

Abstract

Canine distemper virus (CDV) is a negative-sense, single-stranded RNA virus within the genus Morbillivirus and the family Paramyxoviridae. The Morbillivirus genome is composed of six transcriptional units that are separated by untranslated regions (UTRs), which are relatively uniform in length, with the exception of the UTR between the matrix (M) and fusion (F) genes. This UTR is at least three times longer and in the case of CDV also highly variable. Exchange of the M-F region between different CDV strains did not affect virulence or disease phenotype, demonstrating that this region is functionally interchangeable. Viruses carrying the deletions in the M 3' UTR replicated more efficiently, which correlated with a reduction of virulence, suggesting that overall length as well as specific sequence motifs distributed throughout the region contribute to virulence.

Figure 1. Growth characterization of recombinant M-F region exchange mutants.

(A) Schematic drawing of the highly variable region exchanged in recombinant viruses. The recombinant 5804PeH genome is drawn to scale. Black and white boxes represent open reading frames and UTRs, respectively. The genes are indicated by their respective abbreviation. (B) Cell-associated growth curves of the parental and two recombinant viruses in VerodogSLAMtag cells. Cells were infected with a multiplicity of infection (MOI) of 0.01, and samples were harvested daily for five days. Titers are expressed as 50% tissue culture infectious doses (TCID₅₀). Error bars indicate the standard deviation. (C) Syncytia formation in VerodogSLAMtag cells. Cells were infected with a MOI of 0.01 and overlaid with 0.5% methylcellulose. Photographs show a representative syncytia of one replicate taken 48 h post-infection using fluorescence excitation (top panels) and phase contrast (bottom panels) at 100× magnification. (D) Survival curve of ferrets infected with the different mutants. Animals were infected with 10⁵ TCID₅₀ intranasally. Death of an infected animal is indicated by a step down on the graph.

Figure 2. Conserved motifs within the M 3′ UTR.

(A) A pictogram, assembled as described in Materials and Methods, shows the sequence and frequency of the 410 nucleotides of the M 3′ UTR. A base height of 2 bits indicates 100% conservation of that nucleotide across the 26 CDV sequences. The numbering below the pictogram indicates the position in the UTR, with 1 being first nucleotide following the M stop codon and positions 408–410 being the intergenic triplet. (B) Conserved regions within the M 3′ UTR. The M 3′ UTR consists of 410 nucleotides and each is represented by a line. Black lines indicate 100% conservation across the 26 CDV sequences and grey lines represent variable nucleotides.

Figure 3. Generation and characterization of recombinant CDVs with alterations in the M 3′ UTR.

(A) Schematic drawing of recombinant viruses produced. The recombinant 58ΔF₁₀₆ genome is drawn to scale. Black and white boxes represent open reading frames and UTRs, respectively. The genes are indicated by their respective abbreviation. The M-F region of the recombinant viruses is expanded below. The UTRs are indicated by a thinner box, and the signal peptide coding region of the F gene (Fsp) is shaded. (B and C) Growth curves of the parental 58ΔF₁₀₆ and the three recombinant viruses in VerodogSLAMtag cells expressed as titers of released (B) and cell-associated virus (C). Cells were infected with an MOI of 0.01, and samples were harvested daily for five days. Titers are expressed as TCID₅₀ and error bars indicate the standard deviation. **, P<0.01, and ***, P<0.001. (D) Syncytia formation in VerodogSLAMtag cells. Cells were infected with a MOI of 0.01 and overlaid with 0.5% methylcellulose. Photographs show a representative syncytia of one replicate taken 48 h post infection using phase contrast at 100× magnification.

Figure 4. Effects of the M 3′ UTR on transcription and translation.

(A) Northern blot analysis of viral mRNA with DNA probes. VerodogSLAMtag infected with the parental and recombinant viruses at an MOI of 1 were subjected to Northern blot analysis using DIG-labeled probes. Membranes were hybridized with DIG-labeled DNA probes for CDV N, P, M or F. Band intensities were determined by densitometry on non-saturated exposures using the Kodak Molecular Imaging software. The P, M and F expression were normalized by calculating the P/N, M/N and F/N ratios for each virus. The average mRNA ratios are shown beneath the appropriate bands and were calculated from at least four individual experiments. (B) Western blot analysis of viral N, M and F proteins. VerodogSLAMtag cells were infected with the parental and recombinant viruses at an MOI of 1 and overlaid with 0.5% methylcellulose. Cell lysates were extracted after 18 h and subjected to Western blot analysis. The membranes were probed with antibodies specific to the CDV N, M and F proteins. Intensities of N, M and F protein bands were determined by densitometry from non-saturated exposures using the Kodak Molecular Imaging Software. The M and F expression were normalized by calculating the M/N and F/N ratios for each virus and were calculated from at least four individual experiments.

Figure 5. Effects of the M 3′ UTR on early transcription and replication.

Quantitative real-time RT-PCR analysis of viral genome and N, M and F mRNA. VerodogSLAMtag cells were infected with the parental and recombinant viruses at a MOI of 1. RNA was extracted after 0, 4, 16 and 24 h and subjected to reverse transcription. Primer pairs specific to the CDV trailer, N, M and F genes were used. The number of RNA molecules in the sample was quantified by plotting the Ct values against standard curves. Error bars indicate the standard deviation. *, P<0.05, **, P<0.01, and ***, P<0.001.

Figure 6. Characterization of M 3′ UTR mutants in ferrets.

(A) Survival curve of ferrets infected with the different mutants. Animals were infected with 10⁵ TCID₅₀ intranasally. Death of an infected animal is indicated by a step down on the graph. (B) Course of cell-associated viremia displayed as the number of CDV-infected cells per million peripheral blood mononuclear cells (PBMC). (C) CDV-specific IgG response in serum samples over the course of the disease. An immunoperoxidase monolayer assay was performed and antibody titers are displayed as reciprocals of the highest antibody dilution at which viral antigen was observed. Error bars indicate the standard deviation.