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. 2008 Jun;82(12):5933-9.
doi: 10.1128/JVI.02273-07. Epub 2008 Apr 2.

Tumor Antigen LRRC15 Impedes Adenoviral Infection: Implications for Virus-Based Cancer Therapy

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Free PMC article

Tumor Antigen LRRC15 Impedes Adenoviral Infection: Implications for Virus-Based Cancer Therapy

Jim O'Prey et al. J Virol. .
Free PMC article

Abstract

Adenoviruses for gene or oncolytic therapy are under development. Notable among these strategies is adenoviral delivery of the tumor suppressor p53. Since all therapeutics have limitations in certain settings, we have undertaken retroviral suppressor screens to identify genes conferring resistance to adenovirus-delivered p53. These studies identified the tumor antigen LRRC15, which is frequently overexpressed in multiple tumor types, as a repressor of cell death due to adenoviral p53. LRRC15, however, does not impede p53 function per se but impedes adenoviral infection. Specifically, LRRC15 causes redistribution of the coxsackievirus-adenovirus receptor away from the cell surface. This effect is manifested in less adenoviral binding to the surfaces of LRRC15-expressing cells. This discovery, therefore, not only is important for understanding adenoviral biology but also has potentially important implications for adenovirus-based anticancer therapeutics.

Figures

FIG. 1.
FIG. 1.
A screen for suppressors of p53-induced cell death identified LRRC15. (a) Cells containing mutagenizing retroviruses were generated and then challenged with adenoviral p53 as depicted. (b) RT-PCR identified an in-frame viral integration at the beginning of exon 2 of LRRC15. LTR, long terminal repeat; SD, splice donor site; Tet-Off, tetracycline-responsive repressor element; UTR, untranslated region.
FIG. 2.
FIG. 2.
LRRC15 impedes cell death due to adenoviral p53 and decreases p53 levels. (a) ERM-LRRC15 cells were treated with Dox for a period of 48 h prior to infection with adenoviral p53, and the levels of ERM-tagged and total LRRC15 were determined by qPCR. (b) ERM-LRRC15 cells that had been treated with Dox for a period of 48 h were then, where indicated, infected with adenoviral p53 (Ad-p53) or a control “empty” adenovirus (Ad-Cont). Cells were harvested for flow cytometry 48 h postinfection and for Western blotting 24 h postinfection. (c) Saos2 cells were infected with a retrovirus expressing LRRC15 (Saos2-LRRC15) or an empty vector as a control (Saos2-Cont), and the presence of HA-tagged LRRC15 was determined by Western blotting. (d) Saos2-LRRC15 cells and Saos2-Cont cells were infected with Ad-p53 or Ad-Cont and analyzed after 48 h for cell death by flow cytometry and after 24 h for p53 expression by Western blotting. In cell death assays, the percentage of cells with a sub-G1 DNA content was taken as a measure of the extent of apoptosis at the indicated time. NS, not significant; P = 0.12 by a two-tailed t test.
FIG. 3.
FIG. 3.
LRRC15 does not affect p53 stability. Saos2 cells were transiently transfected with the combinations of plasmids indicated. After 24 h, cells were harvested and analyzed for protein expression by Western blotting. Equal amounts of total protein were added to each lane. The results shown are representative of five separate experiments. GFP was included in each transfection as a control for transfection efficiency.
FIG. 4.
FIG. 4.
LRRC15 impedes adenoviral infection. (a and b) After 48 h of pretreatment with Dox (to switch off LRRC15, which is driven by the ERM integration), ERM-LRRC15 cells were infected with an adenovirus containing a GFP transgene (Ad-GFP). Twenty-four hours postinfection, GFP and GFP gene levels were determined by Western blotting (a) and qPCR (b), respectively. MOI, multiplicity of infection (number of virus particles per cell as determined by plaque assay). (c and d) Saos2-LRRC15 cells and control (Saos2-Cont) cells were infected with an adenovirus expressing GFP. Twenty-four hours postinfection, GFP and GFP gene levels were determined by Western blotting (c) and qPCR (d), respectively. (e) HCT-116 and A549 cells were infected either with a retrovirus expressing HA-tagged LRRC15 or with an empty viral vector as a control. The expression of HA-tagged LRRC15 was assessed and compared to that in Saos2-LRRC15 cells by Western blotting. (f and g) HCT-116-LRRC15 and HCT-116-Cont cells were infected with an adenovirus expressing GFP. Twenty-four hours postinfection, GFP and GFP gene levels were determined by Western blotting (f) and qPCR (g), respectively. (h and i) A549-LRRC15 and A549-Cont cells were infected with an adenovirus expressing GFP. Twenty-four hours postinfection, GFP and GFP gene levels were determined by Western blotting (h) and qPCR (i), respectively.
FIG. 5.
FIG. 5.
LRRC15 decreases cell surface, but not total, CAR levels. (a) A549-LRRC15 and A549 control (Cont) cells were analyzed for surface expression of the following parameters, which are known to affect adenoviral transduction: CAR, integrin αv, integrin β3, and integrin β5. Surface expression was quantified by flow cytometry, and the percentage of cells showing a change in surface expression in A549-LRRC15 cells is shown relative to that observed in A549-Cont cells. (b) Total CAR levels in A549-LRRC15 and A549-Cont cells were measured by Western blotting. A lysate from 293 cells is included as a positive-control guideline for detection of CAR expression.
FIG. 6.
FIG. 6.
LRRC15 impedes adenoviral attachment. (a and b) A549-LRRC15 (b) and A549 control (Cont) (a) cells were incubated with Ad-GFP for 90 min on ice to permit adenoviral attachment but not internalization. Cells were subsequently stained with an anti-adenovirus type 5 (Ad5) antibody and 4′,6′-diamidino-2-phenylindole (DAPI) before being visualized by fluorescent microscopy.

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