Human IFIT1 Knockout Cell Line-HEK293T

IFIT is an interferon-inducible protein that binds to the 5'-triphosphate or ribose unmethylated capped ends of mRNAs to inhibit translation. Although some viruses circumvent IFIT by synthesizing RNAs with eukaryotic-like caps, no viral proteins have been identified that antagonize IFIT. Here, researchers show that the N-terminal and C-terminal portions of C9, a protein required for vaccinia virus resistance to the type I interferon-induced state in humans, bind IFIT and the ubiquitin regulatory complex, respectively. The two C9 domains co-target IFIT for proteasomal degradation, thereby conferring interferon resistance similar to that of IFIT knockout. Furthermore, ectopic expression of C9 rescues the interferon sensitivity of a vaccinia virus mutant with an inactive cap-1-specific ribose methyltransferase that is otherwise unable to express early proteins. In contrast, a C9 deletion mutant expresses early proteins but is blocked by IFIT at the subsequent genome uncoating/replication step. Thus, poxviruses exploit mRNA cap methylation and proteasomal degradation to defeat multiple antiviral activities of IFIT.

Studies have shown that the role of IFIT in inhibiting vΔC9 is distinct from that resulting from the lack of ribose methylation of the mRNA cap structure. Previous studies have shown that viral DNA replication is severely inhibited in IFN-treated A549 cells infected with vΔC9. In addition, there is evidence of impaired release of viral DNA from the nucleus. Here, further experiments were performed to assess whether these steps would be rescued in IFN-treated A549 IFIT1 knockout and IFIT3 knockout (KO) cells infected with vΔC9 (Figure 1A). In control mock-infected A549 cells, background I3 staining was not detected and only EdU labeled nuclear DNA (Figure 1B). In contrast to the results obtained with unmodified A549 cells, the I3 and EdU staining patterns of IFIT1 KO cells infected with vΔC9 were not reduced by IFN pretreatment, indicating that IFIT1 is required for the inhibition of genome replication (Figure 1B). Under the same conditions, IFIT3 KO cells were less protected from IFN, while IFIT2 KO cells showed little or no protection from IFN. In AraC-treated cells infected with vΔC9, resistance to IFN-mediated inhibition of uncoating was also greatest in IFIT1 KO cells, followed by IFIT3 and IFIT2 KO cells (Figures 1B and 1C). Compared with A549 cells, the fold increase in genome copy number in IFN-treated and vΔC9-infected IFIT1, IFIT2, and IFIT3 KO cells was approximately 16-fold, 3-fold, and 9-fold, respectively (Figure 1D). Thus, the beneficial effects of IFIT KO on genome uncoating and replication in IFN-pretreated cells were IFIT1 > IFIT3 > IFIT2. Furthermore, inhibition of genome replication could fully explain the reduction of VACV intermediate and late mRNAs and their translation products in vΔC9-infected cells pretreated with IFN.

Figure 1. Restoration of VACV DNA replication in IFN-β-pretreated A549 IFIT1 knockout and IFIT3 knockout cells. (Liu R, et al., 2019)