Figure 2.

Repetition of amiR-pTP and amiR-E1A Coding Units in the scAAV2 Vector Genome Increases Inhibition of hAd5

(A) Determination of hAd5 replication. HeLa cells were infected with hAd5 at an MOI of 0.05 and concomitantly transduced with 1 × 10³ vge/cell of scAAV2-amiR vectors expressing the indicated amiRs. The cells were lysed 48 hr later, and the amount of viral DNA was quantified by real-time PCR. The copy number of each amiR in the vector genome is shown in parentheses. Cells, which were infected only with hAd5, are labeled “hAd5”. Con represents a scAAV2 control vector containing an array of six amiR sequences directed against dsRed. The genome copy number for each sample was related to con ( = 1). Significance was determined compared to con, ***p < 0.001. Significance compared to pTP (1×), #p < 0.05. (B) Determination of viability of hAd5-infected cells. HeLa cells were transduced with 1 × 10⁴ vge/cell of indicated scAAV2-amiR vectors 5 hr prior to infection with hAd5 at an MOI of 1. Cell viability was determined 5 days after infection. Mock, untransduced/uninfected cells ( = 1). Con and hAd5, see under (A). Cell viability for each sample was related to mock infected. Significance compared to con, ***p < 0.001. Significance as shown, ^##p < 0.01 and ^###p < 0.001; n.s., not significant. (C) Relative expression of amiR-pTP and amiR-E1A_2 by scAAV2 vectors. HeLa cells were transduced with 1 × 10³ vge/cell and analyzed 48 hr later for amiR-pTP and amiR-E1A_2 expression. The abundance of amiR-pTP and amiR-E1A_2 expression was analyzed with real-time RT-PCR and corrected for the expression of snU6RNA, *p < 0.05. Results of (A)–(C) show mean values ± SEM.