Cellular transcription factor TFII-I represses adenovirus gene expression

Abstract

To successfully replicate, viruses must overcome innate cellular antiviral responses. With human adenovirus (HAdV), a key viral repressor of cellular antiviral responses is the early protein E4ORF3. E4ORF3 relocalizes numerous cellular antiviral proteins, particularly those involved in the interferon (IFN) and DNA damage response (DDR) pathways, to sequester them from viral replication sites in the nucleus. E4ORF3 also directs SUMO modification of a subset of its targets, some of which are subsequently targeted for proteasomal degradation. We previously identified TFII-I, a cellular transcription factor and DNA repair protein, as one of the proteins most highly SUMOylated by E4ORF3, as well as one of the E4ORF3 degradation targets. In this study, we characterized the effect of TFII-I knockout (KO) on HAdV replication. TFII-I KO significantly increased the infectious virus yield from infected cells, supporting the hypothesis that TFII-I acts as a restriction factor during HAdV infection. TFII-I KO also significantly increased viral genome replication, as well as both early and late gene and protein expression. Our results do not support TFII-I acting as a part of either the DDR or IFN responses during HAdV infection. Our results characterize a novel antiviral function for TFII-I against HAdV that occurs during the early stage of the viral replication cycle and highlight the importance of studying viral countermeasures to the cellular antiviral response, like E4ORF3, to better understand how cells restrict viral infection.IMPORTANCEThe cellular transcription factor TFII-I was previously shown to bind to HAdV late promoters and to E4-mutant viral genomes during replication. More recently, TFII-I was shown to be a degradation target of HAdV protein E4ORF3. Due to the long-established importance of E4ORF3 in countering cellular antiviral responses, this raised the question of whether TFII-I possesses an undiscovered antiviral role against HAdV. It was hypothesized that whether TFII-I played an antiviral role in HAdV infection, it was most likely to be as a repressor of the late transcriptional program. Here, we show the first direct evidence of TFII-I repressing HAdV infection and demonstrate that the inhibitory effect can be detected much earlier in the viral life cycle than previously predicted. Our findings provide insight into the role of TFII-I in the cellular antiviral response.

Keywords: GTF2I; TFII-I; Viral DNA Replication; adenovirus; gene expression.

Fig 1

TFII-I KO does not alter the DDR or IFN response to Ad5 infection. TFII-I KO cells were generated by infecting parental BEC, NBS1-, or HDF cells with a CRISPR-Cas9 and guide RNA containing Lentivirus, followed by clonal selection. TFII-I KO was confirmed via Western blot (A, BEC; C, NBS1-; and G, HDF); α-tubulin or vimentin was measured as loading controls. Two independent TFII-I KO clones were selected for each cell type. (B) Parental and TFII-I KO BECs were infected with E4ORF3^-/E4ORF6^- Ad5 at an MOI of 1, and total cellular DNA was isolated at the indicated times post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3. (D) NBS1^- (NBS/vector), NBS1^- ectopically expressing Nbs1 (NBS/Nbs1), and NBS1^- TFII-I KO cells (NBS TFII-I-/- #8 and #9) were infected with E4ORF3^-/E4ORF6^- Ad5 (dl355/inORF3) at an MOI of 1, and total cellular DNA was isolated at the indicated time post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3. (E) Parental and TFII-I KO BECs were pretreated for 24 h with IFNα. IRF7 levels were measured by Western blot; Vimentin was measured as a loading control. (F) Parental and TFII-I KO HDFs were pretreated for 24 h with IFN-α or IFN-γ and infected with Ad5-WT at an MOI of 1. Total cellular DNA was isolated at the indicated times post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3.

Fig 2

TFII-I KO increases viral yield during Ad5-WT infection. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 1, and cell lysates were collected at 48 (A) and 72 (B) hpi. Viral yields were determined by plaque assay. To generate a multistep growth curve, parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 0.1, and cell lysates were collected at 3 (C), 6 (D), and 9 (E) dpi. Viral yields were determined by plaque assay. The results shown represent the mean values ± SD; n = 3. * =P ≤ 0.05, ** =P ≤ 0.01. The fold increases in virus titers in the multistep growth curves at each time point are shown.

Fig 3

TFII-I KO does not alter viral late gene expression at early times in Ad5-WT infection. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 1. Total RNA was collected at the indicated times post-infection, and the levels of (A) L1 and (B) L4 mRNA were determined by RT-qPCR and normalized to GAPDH. The results shown represent the mean values ± SD; n = 3.

Fig 4

TFII-I KO increases viral late protein and intermediate stage gene expression during Ad5-WT infection. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 10 (A, B) or 1 (C, D). (A, B) Whole-cell extracts were collected at the indicated times post-infection, and the level of Ad5 intermediate and late proteins was determined by Western blot using the indicated antibodies; GAPDH was measured as a loading control. (C, D) Total RNA was collected at the indicated times post-infection, and the levels of IVa2 (C) and pIX (D) mRNAs were determined by RT-qPCR and normalized to GAPDH. The results shown represent the mean values ± SD; n = 6. * =P ≤ 0.05, ** =P ≤ 0.01, *** =P ≤ 0.001.

Fig 5

TFII-I KO increases viral late gene expression during Ad5-WT infection. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 1. Total RNA was collected at the indicated times post-infection, and the levels of L1–L5 mRNAs were determined by RT-qPCR and normalized to GAPDH (A, L1; B, L2; C, L3; D, L4; E, L5). The results shown represent the mean values ± SD; n = 6. * =P ≤ 0.05, ** =P ≤ 0.01, *** =P ≤ 0.001.

Fig 6

TFII-I KO transiently increases Ad5-WT viral DNA replication. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 1, and total DNA was isolated at the indicated time post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 6. * =P ≤ 0.05, ** =P ≤ 0.01.

Fig 7

TFII-I KO increases viral early gene and protein expression during Ad5-WT infection. Parental and TFII-I KO BECs were infected with Ad5-WT at an MOI of 10 (A) or 1 (B, C). (A) Whole-cell extracts were collected at the indicated times post-infection, and the levels of E1A and DBP proteins were determined by Western blot as indicated; GAPDH was measured as a loading control. (B, C) Total RNA was collected at the indicated time post-infection, and the levels of E1A and E2A mRNAs were determined by RT-qPCR and normalized to GAPDH. The results shown represent the mean values ± SD; n = 6. *P ≤ 0.05, **P ≤ 0.01.