Effect of DNA Repair Protein Rad18 on Viral Infection

Host factors belonging to the DNA repair machineries are assumed to aid retroviruses in the obligatory step of integration. Here we describe the effect of DNA repair molecule Rad18, a component of the post-replication repair pathway, on viral infection. Contrary to our expectations, cells lacking Rad18 were consistently more permissive to viral transduction as compared to Rad18(+/+) controls. Remarkably, such susceptibility was integration independent, since retroviruses devoid of integration activity also showed enhancement of the initial steps of infection. Moreover, the elevated sensitivity of the Rad18(−/−) cells was also observed with adenovirus. These data indicate that Rad18 suppresses viral infection in a non-specific fashion, probably by targeting incoming DNA. Furthermore, considering data published recently, it appears that the interactions between DNA repair components with incoming viruses, often result in inhibition of the infection rather than cooperation toward its establishment

Rad18 Affects the Accumulation of Retroviral DNA

<div><p>(A) MEFs Rad18^−/− infected with HIV-1–based EGFP reporter virus present a higher amount of reverse transcription product as compared to wild-type cells. Cells were infected with 12,500 IU of a VSV-G pseudotyped HIV-1 integration-deficient reporter virus for 4 h, and late reverse transcripts were assessed by QPCR at 4, 8, 12, and 24 h after infection.</p><p>(B) Hela cells transfected with Rad18-IRES-HcRed and IRES-HcRed control plasmids were infected with 12,500 IU of a VSV-G pseudotyped HIV-1 integration-deficient reporter virus. HcRed-positive cells were then sorted 12 and 20 h post-infection, and late reverse transcripts were assessed by QPCR.</p></div

Increased Adenovirus Infection in Rad18-Negative Cells

<p>Rad18^−/− MEFs show a greater susceptibility to infection with adenovirus expressing EGFP as a reporter as compared to wild type. Cells were infected with serial dilutions of adenovirus EGFP and assessed by flow cytometry 40–44 h after infection. Virus titers (IU) were measured in MEFs Rad18^−/−.</p

The Increase in Infection of Rad18^−/− Cells Is Independent of Integrase Activity and Is Not Dependent on Illegitimate Integration

<div><p>(A) Rad18^−/− MEFs show a greater susceptibility to infection with an integration-inert HIV-1 virus as compared to Rad18^+/+ cells. Cells were infected with an integration-deficient HIV-1–based EGFP reporter virus using serial dilutions of p24 values equivalent to 125,000 IU of wild-type virus and then assessed after 40–44 h by flow cytometry.</p><p>(B) MEFs Rad18^−/− and Rad18^+/+ infected with an integration-deficient HIV-1–based EGFP reporter virus were assessed by flow cytometry at 2, 5, and 9 d after infection.</p><p>(C) MEFs Rad18^−/− and Rad18^+/+ infected with HIV-1–based EGFP reporter virus and assessed by flow cytometry at 2, 5, and 9 d after infection.</p></div

Rad18 Influences the Susceptibility to Retroviral Infection

<div><p>(A) Rad18^−/− MEFs, demonstrate a greater sensitivity to an HIV-1–based EGFP reporter virus infection as compared to wild type. Cells were infected with serial dilutions of an HIV-1–based EGFP reporter virus and assessed after 40–44 h by flow cytometry. Virus titers (IU) were measured in NIH3T3 cyc.T cells.</p><p>(B) Rad18^−/− MEFs demonstrate a greater sensitivity to infection with MLV vector–derived virus as compared to wild type. MLV-based EGFP reporter virus was used to infect cells that were then assessed by flow cytometry 40–44 h later.</p><p>(C) Hela cells expressing human Rad18 are more resistant to HIV-1 infection as compared to cells transfected with control plasmid. Cells transiently transfected with either hRad18-IRES-HcRed vector or IRES-HcRed control vector were infected with serial dilutions of a HIV-1–based EGFP reporter virus and assessed after 40–44 h by flow cytometry.</p></div

Time to Peak Glucose and Peak C-Peptide During the Progression to Type 1 Diabetes in the Diabetes Prevention Trial and TrialNet Cohorts

OBJECTIVE To assess the progression of type 1 diabetes using time to peak glucose or C-peptide during oral glucose tolerance tests (OGTTs) in autoantibody-positive relatives of people with type 1 diabetes. RESEARCH DESIGN AND METHODS We examined 2-h OGTTs of participants in the Diabetes Prevention Trial Type 1 (DPT-1) and TrialNet Pathway to Prevention (PTP) studies. We included 706 DPT-1 participants (mean ± SD age, 13.84 ± 9.53 years; BMI Z-score, 0.33 ± 1.07; 56.1% male) and 3,720 PTP participants (age, 16.01 ± 12.33 years; BMI Z-score, 0.66 ± 1.3; 49.7% male). Log-rank testing and Cox regression analyses with adjustments (age, sex, race, BMI Z-score, HOMA-insulin resistance, and peak glucose/C-peptide levels, respectively) were performed. RESULTS In each of DPT-1 and PTP, higher 5-year diabetes progression risk was seen in those with time to peak glucose >30 min and time to peak C-peptide >60 min (P < 0.001 for all groups), before and after adjustments. In models examining strength of association with diabetes development, associations were greater for time to peak C-peptide versus peak C-peptide value (DPT-1: χ2 = 25.76 vs. χ2 = 8.62; PTP: χ2 = 149.19 vs. χ2 = 79.98; all P < 0.001). Changes in the percentage of individuals with delayed glucose and/or C-peptide peaks were noted over time. CONCLUSIONS In two independent at-risk populations, we show that those with delayed OGTT peak times for glucose or C-peptide are at higher risk of diabetes development within 5 years, independent of peak levels. Moreover, time to peak C-peptide appears more predictive than the peak level, suggesting its potential use as a specific biomarker for diabetes progression