Research Article: Viral RNA-dependent RNA polymerase mutants display an altered mutation spectrum resulting in attenuation in both mosquito and vertebrate hosts

Date Published: April 4, 2019

Publisher: Public Library of Science

Author(s): K. Lane Warmbrod, Edward I. Patterson, Tiffany F. Kautz, Adam Stanton, Dedeke Rockx-Brouwer, Birte K. Kalveram, Kamil Khanipov, Saravanan Thangamani, Yuriy Fofanov, Naomi L. Forrester, Marco Vignuzzi.


The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.

Partial Text

RNA viruses comprise a diverse group of viruses, which exhibit high genome plasticity due to a high rate of mutation. This results in the generation of a closely related cloud of viral variants known as a ‘quasispecies’ or viral swarm [1]. The generation of this viral swarm is a result of the viral RNA-dependent RNA polymerase (RdRp), which does not possess a proof-reading function. Rather than producing perfect copies of the genome, the RdRp randomly incorporates incorrect nucleotide bases along the genome, generating a cloud of viral genomes that contain one or two mutations in each genome [2]. These variants are believed to collectively contribute to an interactive population that together create the viral phenotype. This population of variants is subject to selection as a whole, rather than selection acting on individual variants. The presence of this diversity is thought to be an advantage for viral transmission and invasion of host tissues [3]. One benefit of adopting a high mutation strategy is that this allows viruses to produce multiple advantageous mutations. The virus is therefore able to infect and replicate in multiple tissue types, each with their own different selective pressures. For arboviruses (arthropod-borne viruses) creating a diverse quasispecies is hypothesized to be of utmost importance, as two diverse species, a vertebrate host and invertebrate vector, must become infected to complete a transmission cycle, and recent work has demonstrated that diversity is important in successful completion of a transmission cycle [4–7].

Viral diversity is an important factor affecting RNA virulence and transmission. Generation of viral diversity allows the virus to effectively move between cells and cell types in vivo. It also increases the potential for the virus to continue its transmission cycle. Virus diversity is thought to be even more important for arboviruses, which infect a two-host system, as the virus must infect both a vertebrate host and a mosquito vector. It is well known that the virus undergoes numerous bottlenecks, or reductions in viral numbers, when going from host to host [6, 20]. This results in a small founding population that initiates infection within the new host. Bottlenecks imposed during transmission decrease the diversity of the viral population, therefore altering the course of infection by inhibiting the pathogen’s ability to adapt to the new host. The smaller the founding population, the slower adaption occurs [21]. A more diverse population covers more sequence space, meaning that the population is more likely to include virus variants required for efficient adaptation. Viruses that are able to produce more diversity with each replication event should be able to replenish diversity in the founding population quickly. Conversely, viruses that are hindered in their ability to generate diversity are hypothesized to be more sensitive to bottlenecks due to the inability to generate a diverse population from the founding population.




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