The DNA repair genes XPB and XPD defend cells from retroviral infection

Abstract

Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in a cellular defense against retroviral infection.

Fig. 1.

Evaluation of transduction efficiency in DNA repair mutant and rescued cell lines. Isogenic mutant and rescued cell lines were transduced with HIV-based and MMLV-based retroviral vectors pseudotyped with VSV-G. The only ORF of the vectors is GFP driven by a CMV promoter leading to expression of GFP after successful integration. The percentage of cells expressing GFP (GFP+) at 48 h was measured by flow cytometry. Each bar represents an individual cell line. (A) From the most NER activity to the least, XPB cell lines are XPB(F99S) fully complemented with the WT XPB allele (XPB-wt), the TTD patient-derived XPB(T119P) mutant, XPB(F99S) complemented with the XPB(T119P) mutant allele (XPB-prt), and the XP/CS patient-derived XPB(F99S) mutant. Paired t test analysis yielded the following two-tailed P values for HIV-GFP infections: XPB-wt and XPB(T119P), P = 0.4; XPB-wt and XPB-prt, P = 0.04; XPB-wt and XPB(F99S), P = 0.01. Analysis of MMLV-GFP infection of XPB-wt and XPB(F99S) yielded P = 0.003. (B) XPD cell lines include two XP patient-derived XPD(R683W) mutant cell lines [XPD(R683W) #1 and XPD(R683W) #2], and each line complemented with the WT XPD allele (XPD-wt #1 and XPD-wt #2). Paired t test analysis yielded the following two-tailed P values: HIV-GFP infections of XPD-wt #1 and XPD(R683W) #1, P = 0.009; XPD-wt #2 and XPD(R683W) #2, P = 0.0002. Analysis of MMLV-GFP infections: XPD-wt #1 and XPD(R683W) #1, P = 0.0006; XPD-wt #2 and XPD(R683W) #2, P = 0.003. (C) XPA cell lines include a patient-derived XPA mutant cell line [XPA(Y116ter)] and complemented with the WT XPA allele (XPA-wt). MSH2 cell lines include WT MEFs (MSH2+/+) and MSH2−/− littermate MEFs. Error bars indicate the standard deviation between duplicate infected wells. An identical trend was observed in at least three separate experiments for all cell lines.

Fig. 2.

Retroviral replication in XP cell lines. Isogenic mutant and complemented XPB (A) and XPD (B) cell lines were infected with an amphotropic murine leukemia virus. Replicating viral production was monitored by RT activity (RT units). Error bars indicate the SD between triplicate infections.

Fig. 3.

Quantitative PCR analysis of HIV cDNA in XPB and XPD cell lines. Isogenic mutant and rescued XPB and XPD cell lines were transduced with an HIV-based retroviral vector pseudotyped with VSV-G. (A and B) Cells were transduced at 20 MOI_293T based on 293T titers. Total DNA extracts were purified at 8, 24, 48, and 72 h. Late reverse transcripts and a cellular genomic marker gene were quantified by qPCR, yielding the number of HIV cDNA molecules per cell. (A) Transductions of XPB(F99S) cells and XPB-wt cells are compared. Paired t test analysis yielded the two-tailed P value for 20 MOI_293T P = 0.0005. (B) Transductions of XPD(R683W) #1 cells and XPD-wt #1 cells are compared. Paired t test analysis yielded the two-tailed P value for 20 MOI_293T, P < 0.0001. The number of 2LTR circles (C) and integrated proviruses (D) per cell were determined by qPCR. Error bars indicate the SD between duplicate infected wells. An identical trend was observed in at least three separate experiments for all cell lines.

Fig. 4.

Evaluation of HIV cDNA degradation after addition of an RT inhibitor. XPB and XPD cell lines were transduced with an HIV-based retroviral vector. Three hours after the addition of the virus, the media was replaced and the RT inhibitor efavirenz (A) or foscarnet (B) was added. The number of cDNA molecules per cell was quantified by qPCR. At each time point, the remaining cDNA (%) was calculated from the number of cDNA molecules per cell treated with efavirenz/foscarnate divided by the number of cDNA molecules per untreated cell. Time indicates the number of hours after the addition of efavirenz. P values associated with efavirenz treatment: XPB-wt and XPB(F99S), P = 0.002; XPD-wt #1 and XPD(R683W) #1, P = 0.01. P values associated with foscarnet treatment: XPB-wt and XPB(F99S), P = 0.001; XPD-wt #1 and XPD(R683W) #1, P = 0.01. An identical trend was observed in at least three separate experiments. Error bars indicate the standard deviation between duplicate infected wells.