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miR-99b and miR-485 Expression Enhances Infective Viral Particle Formation, Viral Release, and In Vitro Cytotoxicity
To evaluate the effects of miR-99b and miR-485 on adenoviral repli-cation, PANC-1 and MIA PaCa-2 Bortezomib (PS-341) were infected with AdwtE miR-99b and AdwtE miR-485 at a low viral dose; after one replication cycle, the concentration of infective viral particles present in the su-pernatant was determined, as stated and at 4 hr after infection to ensure equal viral entry between viruses. Indeed, both miRNAs led to higher numbers of released infective particles compared to AdwtE (Figure 3A). This effect was dose dependent. Differences between AdwtE miR viruses and AdwtE diminished at higher infecting doses (Figure 3B), suggesting a saturation effect, where all the viruses already reached the maximum effects.
To confirm that the increase in released infective particles was specific for miR-99b and miR-485 activity, PANC-1 cells were transfected with miRNA expression plasmids (miRVec-99b or miRVec-485) or a control plasmid (miRVec hTR), followed by AdwtE infection. The release of AdwtE infective particles was increased in the presence of either miRNA (Figure S3A). Furthermore, infection of PANC-1 cells with AdwtE miR-759, a miRNA that remains stable during adeno-virus infection,10,11 resulted in a viral release similar to that of the con-
trol viruses AdwtE and AdwtE hTR (Figure S3B), indicating that the observed effects of candidate miRNAs were specific and not due to universal miRNA effects, such as a block in the miRNA biogenesis pathway.
intracellular Intracellularv.g./ml (AdwtEmiR/AdwtE)
Multiplicity of infection (IFU/cell)
tE miR wtE miR
Figure 3. miR-99b- and miR-485-Encoding Adenoviruses Display Higher Viral Yield, Correlating with Increased In Vitro Oncolytic Activity
(A) Relative infective viral particle release. PANC-1 and MIA PaCa-2 cells were seeded in triplicate and infected (at 0.5 IFU/cell or 5 IFU/cell, respectively) with AdwtE, AdwtE miR-99b, or AdwtE miR-485. At 48 hr PI, supernatants were collected and titrated by viral infectious units. The dashed line represents AdwtE values. (B) Dose dependence effect on infective viral particles released in PANC-1 cells. Cells were seeded in triplicate and infected with AdwtE, AdwtE miR-99b, or AdwtE miR-485 at a range of doses. At 48 hr PI, supernatants were collected and titrated by viral infectious units. The dashed line represents AdwtE values. (C) Time course of intra- and extracellular infective viral particles in PANC-1 cells. Cells were seeded in triplicate and infected with AdwtE, AdwtE miR-99b, or AdwtE miR-485 at a dose of 0.5 IFU/cell. Cells and supernatants were collected at 24, 30, 48, and 72 hr PI and titrated by viral infectious units. (D) Time course of total intracellular viral genome content in PANC-1 cells. Cells were seeded in triplicate and infected with AdwtE, AdwtE miR-99b, or AdwtE miR-485 at a dose of 0.5 IFU/cell. Cells were collected at 24, 30, 48, and 72 hr PI, and the intracellular viral
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We next investigated whether the increase in the number of infective particles in the supernatants was due to an enhanced viral release or to an increase in the number of intracellular particles. PANC-1 cells were infected with AdwtE, AdwtE miR-99b, or AdwtE miR-485, and the amounts of infective viral particles present in the intracellular and extracellular compartments were measured at the indicated time points. Infection with AdwtE miR-99b or AdwtE miR-485 led to a faster release of infective viral particles than did AdwtE. This higher release correlated with a more efficient intracellular content of AdwtE miR infective particles (Figure 3C). In contrast, quantification of intracellular viral genomes showed no differences between viruses at early time points. An increase in the number of intracellular viral genomes was observed at 72 hr post-infection (PI), probably due to the internalization of viral genomes in a second infection cycle (Figure 3D).
The increase in the viral release of AdwtE miR-99b and AdwtE miR-485 led to an enhanced cytotoxic activity, with AdwtE miR-485 leading to a 3-fold decrease in the IC50 values in PANC-1 cells and to a 2-fold decrease in MIA PaCa-2 cells. Similarly, AdwtE miR-99b promoted a 2-fold decrease in the IC50 in both cell lines (Fig-ure 3E). AdwtE miR-485 and AdwtE miR-99b also had significantly reduced IC50 values in CP15-Luc, NP-18, Capan-2, HPAF-II, and Hs766T PDAC cells (Figure S2B). Altogether, these results establish that miR-99b and miR-485 increase the adenoviral fitness through an enhancement in the intracellular content and release of mature virions, thereby conferring increased cytotoxicity to miR candidate-encoding viruses.