Article Figures & Data
Figures
- FIGURE 1.
Construction of mutants and structural analysis of the genomes of reconstituted viruses. A, Schematic representation of the genomes of reconstituted viruses. P indicates the probe used for Southern blot analysis, corresponding to nucleotide positions 102215–103164. B, Southern blot analysis of DNA of reconstituted viruses digested with the restriction enzymes and hybridized with the probe indicated in A. FRT MUT, ORF74FRT mutant; WT, parental virus; STOP MUT, ORF74STOP mutant; REV, ORF74STOP revertant; DEL MUT, ORF74DEL mutant. Marker sizes (in kilobase pairs) are indicated on the left.
- FIGURE 2.
RT-PCR analysis of MHV-68 ORF73, 74, 75c, and M11. NIH 3T3 fibroblasts were infected with wild-type, stop mutant, FRT mutant, or DEL mutant virus at a MOI of 1. Three days later, cells were harvested and RNA was extracted for RT-PCR analysis. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. Markers consisting of a 100-bp DNA ladder (Life Technologies) are shown to the left.
- FIGURE 3.
Growth of ORF74 mutant virus in vitro. A, Single-step growth curves were generated for mutant, wild-type, and revertant virus at a MOI of 1 in NIH 3T3 cells. Samples were frozen at the given times after infection, and the titers were determined by plaque assay on monolayers of NIH 3T3 cells. Similar results were obtained in two independent experiments. B, Multistep growth curves were constructed for mutant wild-type and revertant viruses as described in A, but at a MOI of 0.001. C, Comparison of plaque sizes for mutant, wild-type, and revertant viruses. NIH 3T3 cells were infected with virus and overlaid with medium containing 1.6% CMC. After 6 days of incubation, the cell monolayers were fixed with formaldehyde and stained with crystal violet. Similar results were obtained in five independent experiments.
- FIGURE 4.
Effect of chemokines on MHV-68 replication. A, Dose-response curve for the effect of the CXC chemokine KC on viral replication. Viral titers were determined in the presence or absence of chemokine. Monolayers of NIH 3T3 cells were infected with virus at a MOI of 1 and treated with KC at various concentrations. Production of virus was determined by plaque assay. Data are expressed as mean PFU/ml ± SD from three independent experiments. B, Time course of KC stimulation of MHV-68 replication in NIH 3T3 cells. Monolayers of NIH 3T3 cells were infected with virus at a MOI of 1 and treated with KC (10 ng/ml). Production of virus was determined by plaque assay. Data are expressed as the mean ratio of viral titers in the presence and absence of chemokine ± SD from three independent experiments at 12 and 24 h and two at 48 h. C, Stimulation of viral replication by KC is dependent on the vGPCR. Monolayers of NIH 3T3 cells were infected with wild-type, revertant, Stop, FRT, or Del ORF74 mutant viruses at a MOI of 1 and treated with KC (10 ng/ml). Production of virus 24 h after infection was determined by plaque assay. Data are expressed as the mean ratio of viral titers in the presence and absence of KC ± SD. D, The effect of CC and CXC chemokines on MHV-68 replication. The effect of CC chemokines MCP-1 and RANTES or CXC chemokines MIP-2 and crg-2 (mouse IP-10) on replication of MHV-68 was determined as in C. All chemokines were used at a concentration of 10 ng/ml. Data are expressed as the mean ratio of viral titers in the presence and absence of chemokine ± SD of triplicate determinations from one of two independent experiments that gave similar results. E, Reduction of KC-induced viral replication by crg-2. Monolayers of NIH 3T3 cells were infected in triplicate with wild-type or Stop mutant viruses at a MOI of 1 and treated with KC (10 ng/ml) and/or crg-2 (10 ng/ml). Production of virus 24 h after infection was determined by plaque assay. Data are expressed as the mean PFU/ml ± SD.
- FIGURE 5.
The MHV-68 GPCR signals through a pertussis toxin-insensitive, mitogen-activated protein kinase kinase-dependent, pathway. Monolayers of NIH 3T3 cells were infected with wild-type or Stop mutant viruses at a MOI of 1 in the presence or absence of KC (10 ng/ml). Viral replication was determined 24 h after infection by plaque assay. A, The effect of pertussis toxin (100 μg/ml) on viral replication was determined. B, The effect of PI3-kinase inhibitor LY294002, MEK inhibitors U0126 and PD98059, and control compound U0124 (all added at a concentration of 10 μM) on viral replication was determined. Data are expressed as mean PFU/ml ± SD of triplicate assays from two independent experiments. PI3-kinase and MEK inhibitors caused a significant reduction in the increase in viral replication induced by KC. ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05.
- FIGURE 6.
MHV-68 vGPCR is required for efficient reactivation from latency. A,. Infectious center assay. Mouse splenocytes were infected with wild-type mutant or revertant MHV-68 viruses at a MOI of 1.0 in vitro. After 24 h, splenocytes were plated on fibroblast monolayers and overlaid with CMC. Plaques were counted after 6 days. Data are presented as mean ± SD for triplicate assays in one of two independent experiments performed. In each experiment, there was a highly significant difference in the numbers of infectious centers obtained with wild-type and mutant viruses (∗∗∗, p < 0.0001, in each case). B, Real-time quantitative PCR. Quantitation of viral genomes was performed by real-time PCR with the 5700 Sequence Detection System (PE Biosystems) using primers specific for the MHV-68 gp150 gene. Data are shown for one of two separate experiments performed. Similar results were obtained in each experiment.
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