The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Abstract

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.

FIG. 1.

Enhanced rAAV transduction correlates with MRN degradation by E1b55K/E4orf6. (A) Protein degradation is required for the enhancement of rAAV transduction by E1b55K/E4orf6. WT E1b55K- or GFP-expressing HeLa cells were infected with rAAV.LacZ 24 h before superinfection with rAd.E4orf6 (MOI of 50). MG132 and epoxomicin, or dimethyl sulfoxide, were added at 2 h postinfection for another 16 h. Cells were stained or assayed for β-galactosidase. Quantitation of enzyme activity is shown in the graph below, in which error bars represent standard errors of the means for triplicate samples. inhib., inhibitors. (B) Enhanced transduction correlates with MRN degradation. Stable cell lines expressing GFP, WT E1b55K, or E1b55K mutants were infected with rAAV.LacZ in the presence or absence of rAd.E4orf6 (MOI of 50) for 24 h before β-galactosidase analysis as described for panel A. Enhanced rAAV transduction is observed only with E4orf6 in the WT E1b55K and R240A cell lines, where MRN is degraded.

FIG. 2.

Degradation of MRN correlates with increased rAAV DNA accumulation. (A) WT- or mutant E1b55K-expressing HeLa cells were infected with rAAV.GFP in the presence or absence of rAd.E4orf6 (MOI of 25) for 24 h. Hirt extracted viral DNA was analyzed by alkaline gel electrophoresis and Southern blot hybridization with a GFP probe. The arrowhead indicates a double-stranded form of rAAV DNA in WT E1b55K and R240A cells plus E4orf6, where MRN was degraded. ssDNA (single-stranded DNA) refers to input rAAV DNA. (B) HeLa, WT E1b55K, or H354 cell lines expressing GFP-LacR were infected with rAd.E4orf6 (MOI of 25) for 24 h before superinfection with rAAV.LacO (MOI of 400) for another 16 h. Cells were fixed and analyzed by confocal microscopy for rAAV.LacO foci. Arrowheads show cells with bright foci. (C) Degradation of MRN in GFP-LacR cell lines. HeLa-derived cell lines expressing GFP-LacR or GFP-LacR with E1b55K proteins were analyzed by immunoblotting for E1b55K expression and degradation of the Mre11 complex in the presence of E4orf6. (D) Quantitation of rAAV.LacO foci in E1b55K cell lines. For each treatment, the average for three groups of at least 120 cells each is presented. Error bars represent standard errors of the means.

FIG. 3.

The Mre11 complex limits efficient rAAV transduction. (A) A-TLD 3 cell lines expressing GFP or WT Mre11 were infected with rAAV.LacZ at the indicated MOIs for 48 h before being processed for a quantitative β-galactosidase assay. Infections were performed in triplicate, and error bars represent standard errors of the means (SEM). (B) HCT116 cells or HCT116-derived cells containing shRNA to Rad50 (Rad50KD) were infected in triplicate with rAAV.LacZ at the indicated MOIs and harvested after 48 h for a quantitative β-galactosidase assay and Western analysis. Error bars represent SEM. Statistical analysis was performed using an unpaired Student t test, with asterisks representing a P value of <0.005.

FIG. 4.

WT AAV replication is restricted by the Mre11 complex. (A) WT- or mutant E1b55K-expressing HeLa cells were infected with WT AAV with or without the Ad mutant dl1016 or Ad5 (MOI of 50) for 24 h before being harvested for immunoblot analysis or Hirt DNA extraction and QPCR of viral DNA. QPCR levels were normalized to those in GFP-expressing cells infected with AAV alone. Viral replication and Rep accumulation were greatest in E1b55K cells where the MRN complex was degraded (Ad5 or WT E1b55K and R240A cells with dl1016). (B) AAV replication in E1b55K cell lines by transfection with helper plasmids. HeLa-derived cell lines expressing GFP or E1b55K proteins were transfected with the AAV plasmid pNTC244 in the presence or absence of plasmids expressing E4orf6 and DBP. Infection with Ad5 (MOI of 50) served as a positive control. Viral DNA was extracted from cells after 48 h and analyzed by Southern blot hybridization with a Rep probe. Rfd (replication form dimer), Rfm (replication form monomer), and ssDNA (single-stranded DNA) refer to replicative forms of AAV. (C) NBS cell lines complemented with empty vector (NBS−) or WT Nbs1 (NBS+) were infected with WT AAV in the presence or absence of Ad5 (MOI of 50) or the Ad mutant dl1004 (MOI of 50) for 24 h. Cells were processed for viral DNA and immunoblotting. DNA was subject to Southern blot hybridization with a Rep probe. Rfd, Rfm, and ssDNA refer to replicative forms of AAV as in panel B. Lysates were analyzed by immunoblotting for levels of cellular (MRN) and adenoviral (E1b55K, DBP, and E1A) proteins as shown below.

FIG. 5.

MRN components localize to AAV replication compartments and bind AAV ITRs. (A) MRN localizes to AAV replication centers. HeLa cells were transfected with a DBP expression plasmid with or without the AAV infectious clone pNTC244. Cells were fixed and stained for Rep and DBP to mark AAV centers and for MRN components. (B) AAV replication centers formed by plasmid transfection. Shown are examples of stages of replication centers formed when WT E1b55K or H354 cells were transfected with pNTC244, DBP, and E4orf6 expression plasmids. Cells were stained for Rep and Rad50. Rad50 staining was absent in WT E1b55K cells as it was degraded with E4orf6. (C) Degradation of MRN results in more-advanced AAV replication centers. WT E1b55K- or H354-expressing cell lines were transfected with pNTC244, DBP, and E4orf6 expression plasmids. Rep-positive cells, 200 per treatment, were quantified for replication center stage and expressed as the percentage of cells with centers. (D) Binding of MRN components to the AAV ITR. The AAV ITR was 5′ end labeled, incubated with purified Mre11/Nbs1 proteins (MN), and electrophoresed as described in Materials and Methods. The positions of bound (B) and free (F) ITRs are indicated. sssDNA, sonicated salmon sperm DNA.