Complexities of Viral Mutation Rates.

Many viruses evolve rapidly. This is due, in part, to their high mutation rates. Mutation rate estimates for over 25 viruses are currently available. Here, we review the population genetics of virus mutation rates. We specifically cover the topics of mutation rate estimation, the forces that drive the evolution of mutation rates, and how the optimal mutation rate can be context-dependent.

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4+ T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease.

Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. While bat, camel, and human DPP4 support MERS-CoV infection, several DPP4 orthologs, including mouse, ferret, hamster, and guinea pig DPP4, do not. Previous work revealed that glycosylation of mouse DPP4 plays a role in blocking MERS-CoV infection. Here, we tested whether glycosylation also acts as a determinant of permissivity for ferret, hamster, and guinea pig DPP4. We found that, while glycosylation plays an important role in these orthologs, additional sequence and structural determinants impact their ability to act as functional receptors for MERS-CoV. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and better inform our understanding of virus-receptor interactions associated with disease emergence and host susceptibility.IMPORTANCE MERS-CoV is a recently emerged zoonotic virus that is still circulating in the human population with an ∼35% mortality rate. With no available vaccines or therapeutics, the study of MERS-CoV pathogenesis is crucial for its control and prevention. However, in vivo studies are limited because MERS-CoV cannot infect wild-type mice due to incompatibilities between the virus spike and the mouse host cell receptor, mouse DPP4 (mDPP4). Specifically, mDPP4 has a nonconserved glycosylation site that acts as a barrier to MERS-CoV infection. Thus, one mouse model strategy has been to modify the mouse genome to remove this glycosylation site. Here, we investigated whether glycosylation acts as a barrier to infection for other nonpermissive small-animal species, namely, ferret, guinea pig, and hamster. Understanding the virus-receptor interactions for these DPP4 orthologs will help in the development of additional animal models while also revealing species-specific differences impacting MERS-CoV host range.

A Reverse Genetics Platform That Spans the Zika Virus Family Tree.

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics.IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design.

An Evolving Genetic Architecture Interacts with Hill-Robertson Interference to Determine the Benefit of Sex.

Sex is ubiquitous in the natural world, but the nature of its benefits remains controversial. Previous studies have suggested that a major advantage of sex is its ability to eliminate interference between selection on linked mutations, a phenomenon known as Hill-Robertson interference. However, those studies may have missed both important advantages and important disadvantages of sexual reproduction because they did not allow the distributions of mutational effects and interactions (i.e., the genetic architecture) to evolve. Here we investigate how Hill-Robertson interference interacts with an evolving genetic architecture to affect the evolutionary origin and maintenance of sex by simulating evolution in populations of artificial gene networks. We observed a long-term advantage of sex-equilibrium mean fitness of sexual populations exceeded that of asexual populations-that did not depend on population size. We also observed a short-term advantage of sex-sexual modifier mutations readily invaded asexual populations-that increased with population size, as was observed in previous studies. We show that the long- and short-term advantages of sex were both determined by differences between sexual and asexual populations in the evolutionary dynamics of two properties of the genetic architecture: the deleterious mutation rate ([Formula: see text]) and recombination load ([Formula: see text]). These differences resulted from a combination of selection to minimize [Formula: see text] which is experienced only by sexuals, and Hill-Robertson interference experienced primarily by asexuals. In contrast to the previous studies, in which Hill-Robertson interference had only a direct impact on the fitness advantages of sex, the impact of Hill-Robertson interference in our simulations was mediated additionally by an indirect impact on the efficiency with which selection acted to reduce [Formula: see text].

Glycosylation of mouse DPP4 plays a role in inhibiting Middle East respiratory syndrome coronavirus infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development.

Coronavirus Host Range Expansion and Middle East Respiratory Syndrome Coronavirus Emergence: Biochemical Mechanisms and Evolutionary Perspectives.

Coronaviruses have frequently expanded their host range in recent history, with two events resulting in severe disease outbreaks in human populations. Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2003 in Southeast Asia and rapidly spread around the world before it was controlled by public health intervention strategies. The 2012 Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak represents another prime example of virus emergence from a zoonotic reservoir. Here, we review the current knowledge of coronavirus cross-species transmission, with particular focus on MERS-CoV. MERS-CoV is still circulating in the human population, and the mechanisms governing its cross-species transmission have been only partially elucidated, highlighting a need for further investigation. We discuss biochemical determinants mediating MERS-CoV host cell permissivity, including virus spike interactions with the MERS-CoV cell surface receptor dipeptidyl peptidase 4 (DPP4), and evolutionary mechanisms that may facilitate host range expansion, including recombination, mutator alleles, and mutational robustness. Understanding these mechanisms can help us better recognize the threat of emergence for currently circulating zoonotic strains.

Connecting within-host dynamics to the rate of viral molecular evolution.

Viruses evolve rapidly, providing a unique system for understanding the processes that influence rates of molecular evolution. Neutral theory posits that the evolutionary rate increases linearly with the mutation rate. The occurrence of deleterious mutations causes this relationship to break down at high mutation rates. Previous studies have identified this as an important phenomenon, particularly for RNA viruses which can mutate at rates near the extinction threshold. We propose that in addition to mutation dynamics, viral within-host dynamics can also affect the between-host evolutionary rate. We present an analytical model that predicts the neutral evolution rate for viruses as a function of both within-host parameters and deleterious mutations. To examine the effect of more detailed aspects of the virus life cycle, we also present a computational model that simulates acute virus evolution using target cell-limited dynamics. Using influenza A virus as a case study, we find that our simulation model can predict empirical rates of evolution better than a model lacking within-host details. The analytical model does not perform as well as the simulation model but shows how the within-host basic reproductive number influences evolutionary rates. These findings lend support to the idea that the mutation rate alone is not sufficient to predict the evolutionary rate in viruses, instead calling for improved models that account for viral within-host dynamics.

Cryptic genetic variation can make "irreducible complexity" a common mode of adaptation in sexual populations.

The existence of complex (multiple-step) genetic adaptations that are "irreducible" (i.e., all partial combinations are less fit than the original genotype) is one of the longest standing problems in evolutionary biology. In standard genetics parlance, these adaptations require the crossing of a wide adaptive valley of deleterious intermediate stages. Here, we demonstrate, using a simple model, that evolution can cross wide valleys to produce "irreducibly complex" adaptations by making use of previously cryptic mutations. When revealed by an evolutionary capacitor, previously cryptic mutants have higher initial frequencies than do new mutations, bringing them closer to a valley-crossing saddle in allele frequency space. Moreover, simple combinatorics implies an enormous number of candidate combinations exist within available cryptic genetic variation. We model the dynamics of crossing of a wide adaptive valley after a capacitance event using both numerical simulations and analytical approximations. Although individual valley crossing events become less likely as valleys widen, by taking the combinatorics of genotype space into account, we see that revealing cryptic variation can cause the frequent evolution of complex adaptations.

Dominance effects of deleterious and beneficial mutations in a single gene of the RNA virus ϕ6.

Most of our knowledge of dominance stems from studies of deleterious mutations. From these studies we know that most deleterious mutations are recessive, and that this recessivity arises from a hyperbolic relationship between protein function (i.e., protein concentration or activity) and fitness. Here we investigate whether this knowledge can be used to make predictions about the dominance of beneficial and deleterious mutations in a single gene. We employed a model system--the bacteriophage φ6--that allowed us to generate a collection of mutations in haploid conditions so that it was not biased toward either dominant beneficial or recessive deleterious mutations. Screening for the ability to infect a bacterial host that does not permit infection by the wildtype φ6, we generated a collection of mutations in P3, a gene involved in attachment to the host and in phage particle assembly. The resulting collection contained mutations with both deleterious and beneficial effects on fitness. The deleterious mutations in our collection had additive effects on fitness and the beneficial mutations were recessive. Neither of these observations were predicted from previous studies of dominance. This pattern is not consistent with the hyperbolic (diminishing returns) relationship between protein function and fitness that is characteristic of enzymatic genes, but could have resulted from a curve of increasing returns.

Mouse dipeptidyl peptidase 4 is not a functional receptor for Middle East respiratory syndrome coronavirus infection.

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics.