Combined knockdown of RL13 and UL128 for release of cell-free infectivity from recent HCMV isolates.

Due to strictly cell-associated growth, experiments requiring cell-free virus are not applicable to recent clinical HCMV isolates to date. On the other hand, adaptation to cell-free growth is associated with undesirable changes in the viral gene regions RL13 and UL128. We had previously found that siRNA-mediated reduction of UL128 expression allowed transient release of cell-free virus by clinical isolates, and now hypothesized that virus yield could be further increased by additional knockdown of RL13. Despite the extensive polymorphism of RL13, effective RL13-specific siRNAs could be designed for three recent isolates and the Merlin strain. Knockdown efficiency was demonstrated at the protein level with a Merlin variant expressing V5-tagged pRL13. Knockdown of RL13 alone did not result in measurable release of cell-free virus, but combined knockdown of RL13 and UL128 increased infectivity in cell-free supernatants by a factor of 10-2000 compared to knockdown of UL128 alone. These supernatants could be used in dose-response assays to compare the effect of a neutralizing antibody on the various HCMV isolates. In summary, combined knockdown of RL13 and UL128 by specific siRNAs allows reliable release of cell-free infectivity from otherwise strictly cell-associated HCMV isolates without the need to modify the viral genome.