Response to “HIV Escape From RNAi Antivirals: Yet Another Houdini Action?”

To the Editor: In his 1909 book Handcuff Secrets, Harry Houdini lifted the shroud of secrecy surrounding his performances and revealed that his mystifying capacity to escape shackles and chains was based in painstaking effort and compromise. Shaking off straightjackets for example required dislocating his shoulders, performance after performance, year after year. In the current issue of Molecular Therapy Nucleic Acids, Berkhout and Das argue that our recent findings of indirect HIV escape from RNA interference (RNAi) 1 have been misinterpreted as a " Houdini action " (citation). However, the " escape act " that we observed is not an illusion but a painful additional example of HIV's recurring ability to evade antiviral therapies. In our recent work, 1 we propagated HIV in vitro in the presence or absence of antiviral RNAi for 32 days. Extensive sequencing of virus that escaped RNAi, as well as control virus propagated in parallel cultures for the same period of time in the absence of RNAi, enabled distinction between mutations due to RNAi-mediated selective pressure versus those arising from drift or general selection for replica-tion. Subsequent analysis of over 400 individual sequences revealed a statistically significant increase in nucleotide diversity in the U3 region of the long terminal repeat (LTR) for virus exposed to RNAi. Armed with this in-depth statistical analysis, we then introduced individual, mutant U3 regions back into the original parent HIV strain and clonally tested the roles of these specific LTR mutations on viral replication in the presence or absence of antiviral RNAi. Four such variants showed significantly enhanced replication compared to the wild-type virus in RNAi-protected cells. The RNAi-resistant variants also exhibited enhanced transcriptional activity, and their replicative advantage in RNAi-protected cells was reduced by addition of a RNAi-enhancing small molecule. In concert with our prior investigation, 2 these findings support the hypothesis that HIV can evolve as a stronger promoter to overwhelm the RNAi pathway with a large number of transcripts. Unfortunately, this indirect escape also resulted in cross-resistance to combinatorial RNAi targeting two distinct and spatially distant sites in the HIV genome, thus complicating antiviral RNAi design. In the general spirit of open scientific exchange, we appreciate Berkhout and Das' discussion, and we take this opportunity to discuss a number of questions they raise. One claim is that the mutants found to be indirectly resistant to RNAi are not truly resistant. As shown in Supplementary Figure S3, however, …