Modeling Within-Host Evolution of HIV: Mutation,Competition and Strain Replacement

Virus evolution during infection of a single individual is a well-known feature of disease progression in chronic viral diseases. However, the simplest models of virus competition for host resources show the existence of a single dominant strain that grows most rapidly during the initial period of infection and competitively excludes all other virus strains. Here, we examine the dynamics of strain replacement in a simple model that includes a convex trade-off between rapid virus reproduction and long-term host cell survival. Strains are structured according to their within-cell replication rate. Over the course of infection, we find a progression in the dominant strain from fast- to moderately-replicating virus strains featuring distinct jumps in the replication rate of the dominant strain over time. We completely analyze the model and provide estimates for the replication rate of the initial dominant strain and its successors. Our model lays the groundwork for more detailed models of HIV selection and mutation. We outline future directions and application of related models to other biological situations.     

Quasispecies spatial models for RNA viruses with different replication modes and infection strategies

Empirical observations and theoretical studies suggest that viruses may use different replication strategies to amplify their genomes, which impact the dynamics of mutation accumulation in viral populations and therefore, their fitness and virulence. Similarly, during natural infections, viruses replicate and infect cells that are rarely in suspension but spatially organized. Surprisingly, most quasispecies models of virus replication have ignored these two phenomena. In order to study these two viral characteristics, we have developed stochastic cellular automata models that simulate two different modes of replication (geometric vs stamping machine) for quasispecies replicating and spreading on a two-dimensional space. Furthermore, we explored these two replication models considering epistatic fitness landscapes (antagonistic vs synergistic) and different scenarios for cell-to-cell spread, one with free superinfection and another with superinfection inhibition. We found that the master sequences for populations replicating geometrically and with antagonistic fitness effects vanished at low critical mutation rates. By contrast, the highest critical mutation rate was observed for populations replicating geometrically but with a synergistic fitness landscape. Our simulations also showed that for stamping machine replication and antagonistic epistasis, a combination that appears to be common among plant viruses, populations further increased their robustness by inhibiting superinfection. We have also shown that the mode of replication strongly influenced the linkage between viral loci, which rapidly reached linkage equilibrium at increasing mutations for geometric replication. We also found that the strategy that minimized the time required to spread over the whole space was the stamping machine with antagonistic epistasis among mutations. Finally, our simulations revealed that the multiplicity of infection fluctuated but generically increased along time.     

Mechanisms of genetic robustness in RNA viruses

Two key features of RNA viruses are their compacted genomes and their high mutation rate. Accordingly, deleterious mutations are common and have an enormous impact on viral fitness. In their multicellular hosts, robustness can be achieved by genomic redundancy, including gene duplication, diploidy, alternative metabolic pathways and biochemical buffering mechanisms. However, here we review evidence suggesting that during RNA virus evolution, alternative robustness mechanisms may have been selected. After briefly describing how genetic robustness can be quantified, we discuss mechanisms of intrinsic robustness arising as consequences of RNA-genome architecture, replication peculiarities and quasi-species population dynamics. These intrinsic robustness mechanisms operate efficiently at the population level, despite the mutational sensitivity shown by individual genomes. Finally, we discuss the possibility that viruses might exploit cellular buffering mechanisms for their own benefit, producing a sort of extrinsic robustness.     

Evolutionary Consequences of Mutation and Selection within an Individual

Whether in sexual or asexual organisms, selection among cell lineages during development is an effective way of eliminating deleterious mutations. Using a mathematical analysis, we find that relatively small differences in cell replication rates during development can translate into large differences in the proportion of mutant cells within the adult, especially when development involves a large number of cell divisions. Consequently, intraorganismal selection can substantially reduce the deleterious mutation rate observed among offspring as well as the mutation load within a population, because cells rather than individuals provide the selective ``deaths' necessary to stem the tide of deleterious mutations. The reduction in mutation rate among offspring is more pronounced in organisms with plastic development than in those with structured development. It is also more pronounced in asexual organisms that produce multicellular rather than unicellular offspring. By effecting the mutation rate, intraorganismal selection may have broad evolutionary implications; as an example, we consider its influence on the evolution of ploidy levels, finding that cell-lineage selection is more effective in haploids and tends to favor their evolution.     

Understanding hepatitis C viral dynamics with direct-acting antiviral agents due to the interplay between intracellular replication and cellular infection dynamics

The current paradigm for modeling viral kinetics and resistance evolution after treatment initiation considers only the level of circulating virus and cellular infection (CI model), while the intra-cellular level is disregarded. This model was successfully used to explain HIV dynamics and Hepatitis C virus (HCV) dynamics during interferon-based therapy. However, in the new era of direct-acting antiviral agents (DAAs) against HCV, viral kinetics is characterized by a more rapid decline of the wild-type virus as well as an early emergence of resistant strains that jeopardize the treatment outcome. Although the CI model can be extended to describe these new kinetic patterns, this approach has qualitative and quantitative limitations. Instead, we suggest that a more appropriate approach would consider viral dynamics at the cell infection level, as done currently, as well as at the intracellular level. Indeed, whereas in HIV integrated DNA serves as a static replication unit and mutations occur only once per infected cell, HCV replication is deeply affected by DAAs and furthermore processes of resistance evolution can occur at the intra-cellular level with a faster time-scale.We propose a comprehensive model of HCV dynamics that considers both extracellular and intracellular levels of infection (ICCI model). Intracellular viral genomic units are used to form replication units, which in turn synthesize genomic units that are packaged and secreted as virions infecting more target cells. Resistance evolution is modeled intra-cellularly, by different genomic- and replication-unit strains with particular relative-fitness and drug sensitivity properties, allowing for a rapid resistance takeover.Using the ICCI model, we show that the rapid decline of wild-type virus results from the ability of DAAs to destabilize the intracellular replication. On the other hand, this ability also favors the rapid emergence, intracellularly, of resistant virus. By considering the interaction between intracellular and extracellular infection we show that resistant virus, able to maintain a high level of intracellular replication, may nevertheless be unable to maintain rapid enough de novo infection rate at the extracellular level. Hence this model predicts that in HCV, and contrary to our experience with HIV, the emergence of productively resistant virus may not systematically prevent from a viral decline in the long-term. Thus, the ICCI model can explain the transient viral rebounds observed with DAA treatment as well as the viral resistance found in most patients with viral relapse at the end of DAA combination therapy.     

Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate

Hepatitis C virus (HCV) is present in the host with multiple variants generated by its error prone RNA-dependent RNA polymerase. Little is known about the initial viral diversification and the viral life cycle processes that influence diversity. We studied the diversification of HCV during acute infection in 17 plasma donors, with frequent sampling early in infection. To analyze these data, we developed a new stochastic model of the HCV life cycle. We found that the accumulation of mutations is surprisingly slow: at 30 days, the viral population on average is still 46% identical to its transmitted viral genome. Fitting the model to the sequence data, we estimate the median in vivo viral mutation rate is 2.5×10⁻⁵ mutations per nucleotide per genome replication (range 1.6–6.2×10⁻⁵), about 5-fold lower than previous estimates. To confirm these results we analyzed the frequency of stop codons (N = 10) among all possible non-sense mutation targets (M = 898,335), and found a mutation rate of 2.8–3.2×10⁻⁵, consistent with the estimate from the dynamical model. The slow accumulation of mutations is consistent with slow turnover of infected cells and replication complexes within infected cells. This slow turnover is also inferred from the viral load kinetics. Our estimated mutation rate, which is similar to that of other RNA viruses (e.g., HIV and influenza), is also compatible with the accumulation of substitutions seen in HCV at the population level. Our model identifies the relevant processes (long-lived cells and slow turnover of replication complexes) and parameters involved in determining the rate of HCV diversification.     

Stochastic theory of early viral infection: continuous versus burst production of virions

Viral production from infected cells can occur continuously or in a burst that generally kills the cell. For HIV infection, both modes of production have been suggested. Standard viral dynamic models formulated as sets of ordinary differential equations can not distinguish between these two modes of viral production, as the predicted dynamics is identical as long as infected cells produce the same total number of virions over their lifespan. Here we show that in stochastic models of viral infection the two modes of viral production yield different early term dynamics. Further, we analytically determine the probability that infections initiated with any number of virions and infected cells reach extinction, the state when both the population of virions and infected cells vanish, and show this too has different solutions for continuous and burst production. We also compute the distributions of times to establish infection as well as the distribution of times to extinction starting from both a single virion as well as from a single infected cell for both modes of virion production.     

Phenotypic differences in viral immune escape explained by linking within-host dynamics to host-population immunity

Viruses that do not cause life-long immunity persist by evolving rapidly in response to prevailing host immunity. The immune-escape mutants emerge frequently, displacing or co-circulating with native strains even though mutations conferring immune evasion are often detrimental to viral replication. The epidemiological dynamics of immune-escape in acute-infection viruses with high transmissibility have been interpreted mainly through immunity dynamics at the host population level, despite the fact that immune-escape evolution involves dynamical processes that feedback across the within- and between-host scales. To address this gap, we use a nested model of within- and between-host infection dynamics to examine how the interaction of viral replication rate and cross-immunity imprint host population immunity, which in turn determines viral immune escape. Our explicit consideration of direct and immune-mediated competitive interactions between strains within-hosts revealed three insights pertaining to risk and control of viral immune-escape: (1) replication rate and immune-stimulation deficiencies (i.e., original antigenic sin) act synergistically to increase immune escape, (2) immune-escape mutants with replication deficiencies relative to their wildtype progenitor are most successful under moderate cross-immunity and frequent re-infections, and (3) the immunity profile along short host-transmission chains (local host-network structure) is a key determinant of immune escape.     

Two optimal mutation rates in obligate pathogens subject to deleterious mutation

Pathogen species with high mutation rates are likely to accumulate deleterious mutations that reduce their reproductive potential within the host. By altering the within-host growth rate of the pathogen, the deleterious mutation load has the potential to affect epidemiological properties such as prevalence, mean pathogen load, and the mean duration of infections. Here, I examine an epidemiological model that allows for multiple segregating mutations that affect within-host replication efficiency. The model demonstrates a complex range of outcomes depending on pathogen mutation rate, including two distinct, widely separated mutation rates associated with high pathogen prevalence. The low mutation rate prevalence peak is associated with small amounts of genetic diversity within the pathogen population, relatively stable prevalence and infection dynamics, and genetic variation partitioned between hosts. The high mutation rate peak is characterized by considerable genetic diversity both within and between hosts, relatively frequent invasions by more virulent types, and is qualitatively similar to an RNA virus quasispecies. The two prevalence peaks are separated by a valley where natural selection favors evolution toward the optimal within-host state, which is associated with high virulence and relatively rapid host mortality. Both chronic and acute infections are examined using stochastic forward simulations.     

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.     

Methods of modelling viral disease dynamics across the within- and between-host scales: the impact of virus dose on host population immunity

We study the epidemiology of a viral disease with dose-dependent replication and transmission by nesting a differential-equation model of the within-host viral dynamics inside a between-host epidemiological model. We use two complementary approaches for nesting the models: an agent-based (AB) simulation and a mean-field approximation called the growth-matrix (GM) model. We find that although infection rates and predicted case loads are somewhat different between the AB and GM models, several epidemiological parameters, e.g. mean immunity in the population and mean dose received, behave similarly across the methods. Further, through a comparison of our dose-dependent replication model against two control models that uncouple dose-dependent replication from transmission, we find that host immunity in a population after an epidemic is qualitatively different than when transmission depends on time-varying viral abundances within hosts. These results show that within-host dynamics and viral dose should not be neglected in epidemiological models, and that the simpler GM approach to model nesting provides a reasonable tradeoff between model complexity and accuracy of results.     

INTERGENERATIONAL PHENOTYPIC MIXING IN VIRAL EVOLUTION

Viral particles (virions) are made of genomic material packaged with proteins, drawn from the pool of proteins in the parent cell. It is well known that when virion concentrations are high, cells can be coinfected with multiple viral strains that can complement each other. Viral genomes can then interact with proteins derived from different strains, in a phenomenon known as phenotypic mixing. But phenotypic mixing is actually far more common: viruses mutate very often, and each time a mutation occurs, the parent cell contains different types of viral genomes. Due to phenotypic mixing, changes in viral phenotypes can be shifted by a generation from the mutations that cause them. In the regime of evolutionary invasion and escape, when mutations are crucial for the virus to survive, this timing can have a large influence on the probability of emergence of an adapted strain. Modeling the dynamics of viral evolution in these contexts thus requires attention to the mutational mechanism and the determinants of fitness.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

Dynamic Correlation between Intrahost HIV-1 Quasispecies Evolution and Disease Progression

Quantifying the dynamics of intrahost HIV-1 sequence evolution is one means of uncovering information about the interaction between HIV-1 and the host immune system. In the chronic phase of infection, common dynamics of sequence divergence and diversity have been reported. We developed an HIV-1 sequence evolution model that simulated the effects of mutation and fitness of sequence variants. The amount of evolution was described by the distance from the founder strain, and fitness was described by the number of offspring a parent sequence produces. Analysis of the model suggested that the previously observed saturation of divergence and decrease of diversity in later stages of infection can be explained by a decrease in the proportion of offspring that are mutants as the distance from the founder strain increases rather than due to an increase of viral fitness. The prediction of the model was examined by performing phylogenetic analysis to estimate the change in the rate of evolution during infection. In agreement with our modeling, in 13 out of 15 patients (followed for 3–12 years) we found that the rate of intrahost HIV-1 evolution was not constant but rather slowed down at a rate correlated with the rate of CD4+ T-cell decline. The correlation between the dynamics of the evolutionary rate and the rate of CD4+ T-cell decline, coupled with our HIV-1 sequence evolution model, explains previously conflicting observations of the relationships between the rate of HIV-1 quasispecies evolution and disease progression.     

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.     

Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity

We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSdelta nef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5' end appeared to have less of an effect on infectivity than mutation of the 3' end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3' end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.