Volume 8 issue 12 December 2007
Human Adenovirus Modulates Surfactant Phospholipid Trafficking
Olga L. Miakotina, Diann M. McCoy, Lei Shi, Dwight C. Look, and Rama K. Mallampalli

Supplemental Table 1: Body weight, phospholipids (PL) and protein recovery in control and Ad5-infected animals. C57BL/6 mice received saline or Ad5 (5 x10⁸ pfu/mouse i.t.) at two inoculums on days 1 and 2. 48 h after first exposure to virus, lungs were isolated and used to measure lung mechanics and processed for surfactant DSPC and protein content. Data from 3-5 animals are presented as means ± SEM.

• Independent two-tailed t-test (SPSS).

Supplemental Figure 1: Effect of Ad5 on surfactant-associated proteins. C57BL/6 mice received saline or Ad5 (5 x10⁸ pfu/mouse i.t.) at two inoculums on days 1 and 2. 48 h after exposure to virus, lungs homogenates were isolated to detect surfactant-associated proteins. A. Representative immunoblots detecting surfactant proteins (SP-A, SP-B, SP-C and SP-D) and β-actin in lung homogenates of control and Ad5-treated mice. SP-A was detected as a ~35 kDa band, SP-B – as a ~43 kDa protein, SP-C – as several bands at ~26, 21, 16 and 14 kDa, and SP-D migrated at ~43 kDa. B. Densitometric analysis of immunoreactive bands for surfactant proteins. Results were obtained from 3-4 mice (means ± SEM). #=0.08 vs. controls, t-test.

Figure 1 (.jpg)

Supplemental Figure 2: Apical and basolateral PC efflux at various Ad5 titers. A. Dose-dependence of basolateral PC efflux. Confluent cells on transwells were labeled for 26 h, then washed and infected with replication-deficient d312-Ad5 or wt Ad5 at indicated MOI in the presence of hHDL (40 µg/ml) for 24 h. Medium (apical and basolateral) and cells were analyzed for PC content. Results were obtained in 3 independent experiments (means ± SEM), *p<0.05 vs. respective controls, t-test.
B. Dose-dependence of apical PC efflux. Apical PC efflux was measured as in Fig. 2A in d312-Ad5- or Ad5-infected cells, and results of 3 independent experiments are expressed as the means ± SEM, #p<0.11 vs. respective controls, t-test.

Figure 2 (.jpg)

Supplemental Figure 3: Regulation of apical PC efflux and secretion. A. DIDS does not prevent inhibition of apical PC efflux. Confluent cells on transwells were labeled with [³H]choline (2 µCi/well) for 18 h, then washed, preincubated with 1000 µM DIDS, and infected with Ad5 (MOI=0.5) in the presence of hHDL (40 µg/ml) for 32 h. Medium (apical and basolateral) and cells were analyzed for PC content by TLC. Results were obtained in 3 independent experiments (means ± SEM), *p<0.05 vs. uninfected condition, #p<0.05 vs. DIDS alone, t-test.
B. Ad5 does not affect PKC-mediated PC secretion. MLE cells were plated on plastic and labeled with [³H]choline (5 µCi/2-ml well) overnight, washed and then incubated in FBS-free medium with 0.1 µM TPA, or a diluent (NO TPA) plus or minus Ad5 (MOI=1) for 5 h. Medium and cells were analyzed for PC content. Data are presented relative to controls equal to one, means ± SEM, *p<0.05, n=3, t-test.

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Supplemental Figure 4: Infectivity of human skin fibroblasts. Normal (A) and Tangier disease (B) HSF cells were infected with Ad5-GFP at different MOI for 24 h and photographed (bright field-BF, fluorescence-FL).

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Supplemental Figure 5: Binding of Ad5 to phosphatidylcholine. A, B and C. In vitro binding reaction. 18 µg of Ad5 was incubated with 40 µg of [³H]-labeled DSPC liposomes in 200 µl of HBS for 15 min at room temperature, and reaction fractionated on sucrose gradient. 10 fraction taken were separated on 10% PAGE and probed with rabbit anti-Ad5 antibody for adenoviral capsid proteins (A and B), or counted using scintillation counter (C). Addition of DSPC liposomes to Ad5 in binding reaction caused appearance of adenoviral capsid protein in liposomal enriched fractions (#5 and #6). D and E. In vivo binding. MLE cells were labeled with [³H]choline (10 µCi/10-ml dish) for 24 h, washed and infected with Ad5 at MOI 3 for 16 h. Cells were lysed in hypotonic buffer and fractionated on sucrose gradient. 10 fraction were separated on 10% PAGE and probed with anti-Ad5 antibody for adenoviral capsid proteins (D), or assayed for radioactive PC using TLC (E). In vivo, a significant amount of radioactive PC appeared in fractions containing adenoviral capsid proteins (#9 and #10 fractions, grey area).

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