Final Report Summary - RNAVIRUSDIVNPATHO (The role of genetic diversity of RNA viruses in virulence and pathogenesis)
RNA virus form populations with extreme diversity. Due to their high mutation rates, all RNA virus populations exist as a collection of genetically similar, yet not identical, variants. The existence of RNA viruses as diverse populations was first shown for Qß in 1978 and has now been described for all bacteria, plant and animal RNA viruses (Domingo et al., 2006; Domingo et al., 1978); the term quasispecies, is used to describe the mutant swarms that cluster around a master sequence in these virus populations, that behave like (quasi) a single unit (species). Indeed, when we talk of 'a virus' we refer to a collection of millions of similar variants sharing the same consensus sequence. Much of the sequence heterogeneity of RNA viruses is attributed to the error prone nature of the viral RNA polymerases that replicate them, which lack the proof reading mechanisms of DNA polymerases.
Role of polymerase replication fidelity in RNA virus population diversity. Although polymerase error rate is accepted as one of the key forces driving the high mutation frequencies and evolutionary rates observed in RNA viruses, it has been difficult to determine and separate its contribution from the many other forces that drive mutation rates – such as RNA stability, recombination, and selective pressure. For RNA viruses, a few studies suggest that RNA polymerase fidelity can also be modulated. Higher fidelity reverse transcriptases of HIV, for example, were shown to increase fidelity in vitro and decrease mutation frequencies (Curr et al., 2006). Currently, our work is at the forefront of the study of viral RNA polymerase fidelity and population diversity of RNA viruses in vivo. Our previous studies describing higher fidelity RNA dependent RNA polymerases (RdRp) of poliovirus showed that increases in fidelity, restrict the amount of genetic diversity present in a viral population and compromise a virus' ability to adaptat to new environments (Vignuzzi et al., 2006; Vignuzzi et al., 2008).
Role of RNA virus population diversity in pathogenesis. Although the need to generate diversity of an RNA virus seems intuitive – to adapt to new environments, to escape immune responses, to evolve to higher fitness ; it has been difficult to determine the mechanisms by which diversity is generated and maintained. We showed that a normally diverse population is pathogenic and that restricting population diversity attenuated the virus. We also showed that a wild type population could cooperate with a restricted population to restore pathogenicity and viral dissemination (Vignuzzi et al., 2006). Until the work performed here, only the poliovirus model had been used to study these phenomena in vivo (Pfeiffer and Kirkegaard, 2006; Vignuzzi et al., 2006). Since the publication of these studies, more groups have become interested in developing such models. An FMDV polymerase variant with decreased sensitivity to RNA mutagens (possibly higher fidelity) was isolated (Sierra et al., 2007), and a Coronavirus mutant with increased mutation frequency was also described (Eckerle et al., 2007), that opened these studies to a larger group of viruses.
Diversity-based attenuation of vaccines? The observation that restricting genetic diversity of a poliovirus population by increasing fidelity brings forth the question of whether this approach could constitute a new strategy to attenuate viruses for live vaccine development. Since the first demonstration of a direct link between the restriction of RNA virus population diversity and pathogenesis/attenuation is relatively new (Vignuzzi et al., 2006), its implications in vaccine development will not be studied in detail for some time. In the poliovirus mouse model of infection, we showed that fidelity based virus attenuation constitutes an novel approach to developing live virus vaccines (Vignuzzi et al., 2008).
Coxsackie virus as a new model to study RNA virus populations. To continue out studies in this proposal, we extended our work into a new virus model, Coxsackie virus B2. The Coxsackie viruses are the leading cause of viral myocarditis in humans and Coxsackie B3 virus (CVB3) relies on a common surface receptor naturally present in both humans and mice, making it a better suited study model in the lab (Bergelson et al., 1997; Bergelson et al., 1998).
The overall goal of this proposal was to examine the significance of genome diversity for virus pathogenesis. Specifically, we proposed:
Aim 1: To characterize the relationship between polymerase fidelity and the generation of the viral quasispecies.
Aim 2: To test the suspected correlation between genetic diversity and pathogenesis in more natural models of virus infection in mice.
Aim 3: To develop a new vaccine attenuation strategy based on the modulation of viral population diversity and polymerase fidelity
Thanks to the work performed here, we identified the first high and low fidelity variants for Coxsackie virus (Levi et al., PLOS Pathogens, 2010), that also allowed us to discover a previously unknown mutagenic, antiviral activity for amiloride compounds. We also were able to characterize the first described mutator (low fidelity) strains for an RNA virus in vivo, showing that lowering replication fidelity will also result in virus attenuation. This work was also significant in demonstrating that nature has indeed fine-tuned replication fidelity to strike a balance between adaptability and keeping genome integrity (Gnädig et al., PNAS 2012). Overall, we were able to confirm the correlation that exists with altering fidelity and genetic diversity and virus fitness in vivo: the farther from the wild type level that fidelity is increased or decreased, the more the level of attenuation. Finally, our demonstration that altering fidelity results in attenuation and impedes the establishment of a persistent infection (a common problem for CVB3 that leads to heart disease), underscored the potential of using these approaches to develop safer vaccines. based on these observations, we developed new methods of changing the genetic diversity and adaptability of CVB3, which also result in virus attenuation and new potential strategies to develop vaccines.