Reply to Qu et al., “Quasispecies are constantly selected through virus-encoded intracellular reproductive population bottlenecking”

REPLY

We appreciate the careful reading by F. Qu, K. Khemsom, C. Perdoncini Carvalho, and J. Han of our minireview on SARS-CoV-2 quasispecies recently published in the Journal of Virology (1). Qu and colleagues disagree with one of our assertions about viral quasispecies in general, namely that the mutant genome copies generated during viral RNA replication can collectively engage in evolution. Their main argument is that when a viral population enters a cell, it becomes strongly bottlenecked, and only one or a few of the viruses have a chance to replicate. The remaining copies are degraded, although they may contribute to the cellular modifications required for the formation of replication organelles. An advantage of such bottlenecking for virus survival is that genomes that, during prior replication, mutated to encode a different phenotype find an unchallenged cellular environment to feely multiply and express the deviant phenotype. This model is termed “Bottleneck, Isolate, Amplify, Select” (BIAS), and it is presented as contrary to the premise of quasispecies theory because “the BIAS model is unapologetically deterministic and predicts natural selection as the primary driver of virus evolution.”

We accept the importance of bottlenecks in virus multiplication and spread, but we disagree that such bottlenecks at the stage of virus entry into the cell eliminate the possibility of intra-mutant spectrum interactions. Next, we provide specific examples of the coexistence of mutant and recombinant viral genomes within an infected cell and interactions as modulators of the behavior of the population ensemble. In several cases, interactions have proven the source of new phenotypes.

There were early indications that viral mutant clouds did not behave as mere aggregates of mutants. For example, biological clones of RNA viruses display lower replicative fitness than the parental population, and the population ensemble can suppress specific viral phenotypes (in studies with vesicular stomatitis virus, poliovirus, foot-and-mouth disease virus, and lymphocytic choriomeningitis virus) (1, 2). These evidences were followed by reports of cooperation among measles virus (MeV) mutants for membrane fusion, enhancement of hepatitis B virus replication by coexisting variants, complementation in West Nile virus, and cooperation between two neuraminidase mutants of influenza virus [references and additional examples of modulation by standard and defective genomes can be found in references (2, 3)]. At least some of the molecular mechanisms that have been identified as underlying such interactions (formation of mosaic capsids or multimeric proteins with mutant and wild-type protein subunits) necessitate the coexistence of mutants in the same cell (3). Mutant or recombinant forms of vesicular stomatitis virus, influenza virus, or human immunodeficiency virus type 1 have been identified as coexisting with wild-type genomes in individual cells (4–6).

Shirogane and colleagues (3) divided the types of interactions that occur within a virus population into cooperation, interference, and complementation and reported several examples, as we have also reviewed in previous articles [i.e., reference (7)]. MeV hemagglutinin (H, a tetrameric protein) and the fusion protein (F, a trimeric protein) are involved in cell-to-cell fusion and syncytia formation. Shirogane et al. (3), showed that cooperation between subunits of oligomeric F produced a new phenotype. Wild-type F and mutant F (with G264R) together, but not individually, could mediate membrane fusion in conjunction with a modified H (H-tag). F heterotrimers exhibited enhanced fusion activity with wild-type H as compared with F homotrimers. Viruses containing mixed genomes [wt H/wt F + wt H/F(G264R)] spread in the hamster’s brain, while the virus wt H/wt F did not. In this case, cooperation might be promoted by polyploidy, which is well established for this virus group. Cooperation based on heterooligomer formation inside an infected cell has also been described for influenza virus, as reviewed by Shirogane and colleagues (3).

In another study, Shirogane et al. described hyperfusogenic MeV mutant F proteins that likely enabled cell-to-cell fusion at synapses, as well as “en bloc” virus transmission between neurons (8). The results suggest the coexistence of wild type and mutant genomes in the early phase of MeV persistence in the brain.

Yousaf et al. (9) detected the presence of two MeV genome subpopulations in the brain specimens of a patient with subacute sclerosing panencephalitis, a MeV-related human brain disease. Single cells were analyzed by an allele-specific amplified fluorescence in situ hybridization that detected the presence of the two genome types in many individual cells (9). In this case, genetic complementation may have enhanced viral replication.

“En bloc” transmission of viruses in cellular vesicles delivers multiple particles into the same cell, resulting in enhanced viral replication. Evidence has been reported with enteroviruses (10) and other virus-host systems (11). “En bloc” transmission constitutes a widespread means to introduce multiple particles into a cell, capable of eluding super-infection exclusion, resulting in a more efficient infection than that by individual particles (11).

RNA recombination and genome segment reassortment of segmented genomes, extensively documented with animal and plant RNA viruses, require that a cell be infected by at least two parental viruses. Among many studies with influenza virus on the formation of reassortant progeny, in the one quoted by Qu and colleagues (12), reassortment was quantified using barcoded variant viruses of a pandemic H1N1 strain. Reassortment was pervasive in different hosts, and it was qualified as a diversification process shaped by spatial compartmentalization. When superinfection exclusion operates, there is a time interval (dependent on the virus-host system) in which the exclusion is not yet in operation and successive particles can enter the same cell.

The mutation rates that have been calculated for bacterial, plant, and animal RNA viruses suggest the production of variant genomes within an infected cell. The capacity to sustain viral replication varies among individual cells in culture and within tissues and organs (2). For example, subsets of cultured cells infected with SARS-CoV-2 produced around 10⁵ viral RNA molecules per cell (13). In some cases, cellular editing activities can be an additional source of mutations. Thus, there is ample opportunity for the production of variant genomes during intracellular viral replication. This is also reflected in the stochastic generation of recombinants, with fitness differences guiding the dominance of some recombinant forms over others (14, 15). We discussed this example in our minireview (1) in the context of stochastic events that pervade virus evolution and spread, in which bottlenecks of different intensities play a salient role.

We appreciate the value of the BIAS model because it may indeed operate to limit the replication of multiple virus variants in the same cell. However, the evidence of intra-mutant spectrum interactions within a cell is overwhelming. In its absence, it would be difficult to explain the origin of recombinant and reassortant viruses, observed with animal and plant viruses (16).

We do not see the BIAS model as opposing or rivaling the conceptual body offered by quasispecies theory, which is rooted in Darwinian evolution and information theory. BIAS can be regarded as an interesting complement that emphasizes the consequences that a type of bottleneck taking place at the step of virus entry into the cell can have for the survival of some deviant phenotypes. We see it as a facet of the effects of bottlenecks that we qualified as “weakening, liberation, or challenge” in a previous article (7). The BIAS model raises the interesting possibility that a balance is achieved between “collective modulation” and “productive isolation”, similar to a possible balance between individual and group selection (1, 2). How frequently multiple variants are produced within infected cells should become clearer when the methodology of single-cell ultra-deep sequencing RNA analysis allows the detection of the novo mutations when viral RNA replication is ongoing (17).

A precision. Quasispecies theory was formulated by Eigen and Schuster. Independently, Domingo, working with Weissmann, calculated a mutation rate for bacteriophage Qβ and obtained experimental evidence of quasispecies dynamics for this bacterial virus. With his group, he then extended the concept and implications to animal RNA viruses (18).