Fig. 5: The antiviral role of IFIT1 and Mx1 against HCMV.

a HFFs were silenced for IFIT1 and Mx1 using specific siRNAs (siIFIT1, siMx1, respectively). As negative control cells were also similarly transfected with scrambled siRNA (siCTRL). At 24 hpt, cells were infected with HCMV at an MOI of 0.1 PFU/cell. Viral supernatants were collected at 144 hpi and analyzed by standard plaque assay. Values are expressed as means ± SEM of three independent experiments (siCTRL vs. siIFIT1 P = 0.0111, unpaired two-tailed t test; siCTRL vs. siMx1 P > 0.05, unpaired two-tailed t test). b HFFs were transduced with AdVIFIT1 or AdVLacZ at an MOI of 10 PFU/cell. Subsequently, cells were infected with HCMV at an MOI of 1. The extent of virus replication was measured at 144 hpi. Results are expressed as means ± SEM of three independent experiments (AdVLacZ vs. AdVIFIT1 P > 0.05, unpaired two-tailed t test). c Gel electrophoretic mobility shift assay of IFIT1 binding to PPP-7SKas RNA. 50 nM of in vitro transcribed 7SK 5’-ppp-RNA was incubated with only buffer (buffer), 5 µM of recombinant IFIT1, either untreated (UNT rIFIT1) or treated with PAD2 (PAD2’d rIFIT1) and loaded on a tris-glycine agarose gel. Data are representative of three experiments. d Long-chain DNA synthesis directed by UL54 in the presence or absence of purified UL44ΔC290 was assayed by measuring the incorporation of labeled [₃₂P]TTP with a poly(dA)-oligo(dT) primer template. DNA products were resolved on an alkaline agarose gel that was exposed to film and, for quantification, to a phosphorescence screen followed by scanning with a Typhoon scanner. The image shows products directed by UL54 alone, lane 1), or by UL54 in the presence of untreated (UNT) UL44ΔC290 (lane 2), in the presence of UL44ΔC290 treated with PAD2 or PAD4 (PAD2’d, PAD4’d; lanes 3 and 4, respectively), in the presence of UNT UL44ΔC290 but with 270 pM PAD2 (lane 5) or 370 pM PAD4 (lane 6) added to the DNA synthesis reaction, or in the presence of UL44 incubated in PAD reaction buffer, but without PADs (mock-PAD, lane 7). One representative gel of three independent experiments is shown. Data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.