In 1971, Manfred Eigen mused on the biological chicken-and-egg problem1. In his deliberations, he wondered whether information (encoded in nucleic acids) followed function (executed by proteins) or vice versa. Eigen proposed an original mathematical framework that tried to encompass and explain the “self-organization of matter and the evolution of macromolecules” – in light of physicochemical principles and Darwinian evolutionary theory. Based largely on experimental evolution experiments by Sol Spiegelman using Qb phage RNA replication2, he concluded that “selective advantage is always related to the reproduction of the whole species or ensemble”1. This initial theoretical concept is now known as the foundation of quasispecies theory, although the term was only introduced in a subsequent series of theoretical papers3-5.
Originally focusing on the macromolecule RNA, which some viruses chose as their carrier of genetic information, the theory gained traction when the organisms as a whole were analyzed. A number of viruses were shown to behave in ways commensurate with quasispecies predictions6-8, where viruses exist as a “complex, self-perpetuating population of diverse, related entities that act as a whole”9. A controversy began to emerge between traditionalists on the one hand contending that RNA virus dynamics could be explained by standard population genetics and proponents of quasispecies theory10. The latter predicts that (RNA) virus genomes form a cloud of closely related sequences that are present in a replicative environment at mutational equilibrium and at frequencies that reflect their individual fitness. Although the debate is resolved from a theoretical standpoint11, its relevance to real-world viruses has remained a topic of ongoing debate12, not least because selection in quasispecies operates on the population as a whole, the “ensemble”, rather than at the level of single, individual genomes. In this respect, the debate has often lacked a clear definition of the term “individual”, which does not apply to viruses in the same way as to pro- or eukaryotic organisms13 due to the fact that viruses lack a coherent physiological entity such as a cell and can interact much more intimately with each other.
In our paper, we experimentally augmented genomic variability of a large DNA herpesvirus, an unusual choice for addressing the concept of quasispecies. Marek’s disease virus (MDV) is an oncogenic and deadly virus of the domestic chicken. Starting in the late 1960’s, vaccination has successfully controlled the disease, albeit at the cost of constantly increasing virulence of circulating field strains14. We modified the viral DNA polymerase such that it lost the ability to efficiently perform proofreading, an essential function of all replicative DNA polymerases, which removes erroneously incorporated bases and significantly contributes to DNA replication fidelity. Increasing error rates 80-fold – from about 0.5 per passage of the virus with its 180,000 bp genome to 40 or more – resulted in the so-called error catastrophe and caused the collapse of the virus populations. However, increasing population sizes resulted in viable virus progeny that, as we concluded, then adopted a behavior consistent with quasispecies theory. Virus populations grew in cultured cells with wild-type like kinetics and maintained high genetic diversity across multiple generations. Intriguingly, most individual virus clones were significantly less fit than the quasispecies-like population as a whole. This suggested that selection might indeed be operating on the entire virus population and that the virus stably maintained diversity with virtually no detectable selection of advantageous mutations. Perhaps most surprising of all, the quasispecies-like populations were more virulent than wild-type in the natural host. Importantly, we are able in the herpesvirus system, to defuse one argument brought forward in the case of RNA virus variability, namely that high mutational loads are not proof of quasispecies but rather a corollary of high replication speed. In our engineered MDV, error frequency can be decoupled from DNA polymerase processivity, meaning that error frequency can be investigated separately from the speed of DNA replication.
The controversy surrounding virus quasispecies behavior is primarily due to the limitations of currently available experimental systems, particularly in relation to the exploration of sequence space by viruses starting with a single master sequence. Semantics may also interfere with a more sober debate as there are core properties of virus populations that everyone agrees on. For example, the remarkable diversity expressed by many RNA viruses is beyond doubt. Furthermore, population genetics theory is robust in predicting quasispecies effects under the appropriate conditions. The debate will continue to swirl around whether the appropriate conditions are indeed met in the real world. We hope that our system of artificially engineering a large DNA virus to behave more like an RNA virus can be useful for gaining more insight into the origins and molecular evolution of viruses.
Figure: The illustration schematically shows that wild-type viruses have very uniform genomes in vivo and in vitro, which occupy a very small sequence space (left panel). In contrast, proofreading-impaired quasispecies viruses sample a much larger sequence space (modified according to Eigen, 19939).
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