The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection
Figure 6
ORF54-null virus has a moderate defect in the presence of type I IFN signaling.
A) In these multiple-step growth curves, NIH3T3 or Vero cells were infected at MOI 0.01 and harvested for 5 consecutive days post infection. Viral titers were calculated by plaque assay following three freeze and thaw cycles of the whole cell and supernatant lysate. This data is one representative from 3 independent trials. NIH3T3 curves are shown in the left panel, Vero curves in the right. B) Bone marrow derived macrophages isolated from IFNα/β receptor knock-out mice and wild-type controls were infected with each indicated virus at MOI 4. 60 hpi, cells were harvested and immunoblotted for viral late antigen ORF65. The blots were stripped and re-probed for β-actin. UI = uninfected; 54S = 54Stop; DM = 54DM. C) Infection was completed as described in B. At 60 hpi, cells were harvested and viral titers were calculated by plaque assay following three freeze and thaw cycles of the whole cell and supernatant lysate. The assay was done in triplicate; averages are shown here with error bars reflecting one standard error. For 54Stop compared to WT, 54DM, and 54R, p-values are 0.0004, 0.003, 0.0007, respectively. D) NIH3T3 cells were left alone or pretreated with 100 units/mL of IFN-α one day prior to infection by each indicated virus at MOI 0.01. Immediately following and 2 days post infection, IFN-α was supplemented into the media at 100 units/mL in the treated samples. On 4 days post infection, infected samples were harvested and subjected to three freeze and thaw cycles prior to titer by plaque assay. The assay was done in triplicate; averages are shown here with error bars reflecting one standard error. For 54Stop compared to WT, 54DM, and 54R, p-values are 0.0007, 0.003, 0.008, respectively. For 54Stop, the p-value comparing untreated and IFN-α treated cells is 0.022.