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Reply to Karapetyan: Viral synthesis and assembly is unlikely to occur under cell-free PMCA conditions

The letter to the editor by Karapetyan (1) takes issue with our conclusion in PNAS (2) that the generation of transmissible spongiform encephalopathy (TSE) infectivity in cell-free conditions is against the concept that the TSE infectious agent is a virus. Karapetyan's main point is that poliovirus has been shown to “replicate in a cell-free system” by Wimmer and colleagues (3). We would disagree with the interpretation that these poliovirus experiments are analogous to our protein misfolding cyclic amplification (PMCA) reactions. The divalent cations (Mg2+) and energy sources (ATP and GTP) required for synthesis of viral proteins and nucleic acids, as well as virus assembly (3, 4), should not be present at appropriate concentrations in the crude 1% Triton X-100 brain homogenates made in PBS with EDTA that were used for PMCA reactions. Furthermore, in scrapie PMCA experiments, we and others were able to passage and amplify infectivity serially over multiple reactions, whereas in the poliovirus experiments, serial cell-free passage of the assembled infectious viruses was not demonstrated and would probably not be possible without a mechanism for gently uncoating the viral RNA packaged within the particles. For all these reasons, the generation of new scrapie infectivity in the amounts we detected in PMCA reactions (up to 3 × 105 LD50/0.050 mL reaction mix) is not likely to represent viral synthesis and assembly into infectious TSE virus particles.