Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background

Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.

Principal Findings

After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.

Conclusions

Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.     

Duration and fitness dependence of quasispecies memory.

The duration and fitness dependence of memory in viral quasispecies evolving in cell culture have been investigated using two genetic markers of foot-and-mouth disease virus (FMDV). In lineages of antigenic variant FMDV RED, which reverted to FMDV RGD, memory FMDV RED genomes were detected after 50 infectious cycles, and memory level was fitness dependent. In growth-competition experiments between a reference FMDV RGD and two different FMDV RED populations, a 7.6-fold higher fitness of the initial FMDV RED population resulted in 30 to 100-fold higher memory level. In lineages of low-fitness clones containing an elongated internal polyadenylate tract, revertants lacking excess adenylate residues became dominant by passage 20. However, genomes including a larger number of adenylate residues were detected as memory genomes after at least 150 infectious cycles. Thus, quasispecies memory can be durable and is fitness dependent, as predicted from the growth competition of two mutant forms of a genome. An understanding of factors influencing quasispecies memory levels and duration may have implications for the extended diagnosis of viruses based on the quantification of minority genomes.     

Viral fitness can influence the repertoire of virus variants selected by antibodies

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.     

Fitness increase of memory genomes in a viral quasispecies

Viral quasispecies may contain a subset of minority genomes that reflect those genomic sequences that were dominant at an early phase of quasispecies evolution. Such minority genomes are referred to as memory in viral quasispecies. A memory marker previously characterized in foot-and-mouth disease virus (FMDV) is an internal oligoadenylate tract of variable length that became dominant upon serial plaque-to-plaque transfers of FMDV clones. During large population passages, genomes with internal oligoadenylate were outcompeted by wild-type revertants but remained in the mutant spectra as memory genomes. Here, we report a quantification of relative fitness of several FMDV clones, harboring internal oligoadenylate tracts of different length, and that were retrieved at early or late times (passage number) after implementation of memory. The results show that for any given length range of the oligoadenylate, maintenance in memory resulted in an increase in relative fitness, comparable to the increase undergone by the entire population. The fitness increase is in agreement with the Red Queen hypothesis, and implies a replicative memory mechanism. Thus, permanence of memory genomes may be a source of high fitness variants despite their initial low fitness, and despite having remained hidden in mutant spectra. This reinforces the interest of diagnosing minority genomes during chronic human and animal viral infections.     

Quasispecies Theory and the Behavior of RNA Viruses

A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy.     

Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.     

Viral population dynamics and virulence thresholds

Viral factors and host barriers influence virally induced disease, and asymptomatic versus symptomatic infection is governed by a 'virulence threshold'. Understanding modulation of virulence thresholds could lend insight into disease outcome and aid in rational therapeutic and vaccine design. RNA viruses are an excellent system to study virulence thresholds in the context of quasispecies population dynamics. RNA viruses have high error frequencies and our understanding of viral population dynamics has been shaped by quasispecies evolutionary theory. In turn, research using RNA viruses as replicons with short generation times and high mutation rates has been an invaluable tool to test models of quasispecies theory. The challenge and new frontier of RNA virus population dynamics research is to combine multiple theoretical models and experimental data to describe viral population behavior as it changes, moving within and between hosts, to predict disease and pathogen emergence. Several excellent studies have begun to undertake this challenge using novel approaches.     

RNA replication kinetics, genetic polymorphism and selection in the case of the hepatitis C virus

We show in a simple theoretical quasispecies model that the replication dynamics of hepatitis C virus and a related model-system, the bovine viral diarrhoea virus, result in an effective reduction of RNA templates in infected cells. Viral fitness does not translate directly into RNA sequence replication efficiency, and hence the abundance of the viral master sequences diminishes over time. Our results suggest that genes not involved in RNA replication accumulate mutations over time because they do not undergo selection during this phase. The selection of viral RNA occurs not only during replication but also during the ensuing stages of the viral life cycle: (i) envelopment of viral RNA and (ii) successful infection of other cells, which also requires functionality of non-replicative genes. In particular, viral fitness requires the ability of the genome to encode structural proteins which do not encounter selective pressure during RNA replication. We conclude by discussing the potential value of antiviral drugs which inhibit selection on parts of the viral genome.     

Adaptation of Puumala hantavirus to cell culture is associated with point mutations in the coding region of the L segment and in the noncoding regions of the S segment

We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A-->G and U-->C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.     

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.     

Fitness landscape of human immunodeficiency virus type 1 protease quasispecies

Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected na?ve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.     

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses.

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.     

Antigenic stimulation specifically reactivates the replication of archived simian immunodeficiency virus genomes in chronically infected macaques

Human immunodeficiency virus/simian immunodeficiency virus (SIV) diversification is a direct consequence of viral replication and occurs principally in secondary lymphoid organs where CD4(+) T cells are activated and proliferate. However, the evolution of viral quasispecies may also be driven by various nonexclusive mechanisms, including adaptation to specific immune responses and modification of viral fitness. Analysis of viral quasispecies in SIV-infected macaques subjected to repeated antigenic stimulations allowed us to demonstrate transient expansions of SIV populations that were highly dependent upon activation of antigen-specific T cells. T-cell clones expanded in response to a particular antigen were infected by a specific viral population and persisted for prolonged periods. Upon a second stimulation by encounter with the same antigen, these specific genomes were at the origin of a new burst of replication, leading to rapid but transient replacement of the viral quasispecies in blood. Finally, longitudinal analysis of SIV sequence variation during and between antigenic stimulations revealed that viral evolution is mostly constrained to periods of strong immunological activity.     

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.     

Red queen dynamics, competition and critical points in a model of RNA virus quasispecies.

RNA viruses offer a unique opportunity for the study of evolution at the molecular level. Recent experiments involving clonal populations of RNA viruses have shown that competition among virus strains of approximately equal relative fitness can result in the eventual competitive exclusion of one of the species. As competition proceeds in time, both the winners and the losers exhibited absolute gains in fitness, consistent with the "Red Queen" hypothesis of evolution. Further experiments involving closely related evolving quasispecies revealed a highly predictable nonlinear behavior suggesting a deterministic component in the underlying quasispecies dynamics. This is apparently in contradiction with the standard view of RNA virus evolution as a highly unpredictable process. In this paper we present a simple model which allows previous hypothesis to be tested and provides an interpretation for the observed experimental results.     

Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

Background  

Comparison of data produced on different microarray platforms often shows surprising discordance. It is not clear whether this discrepancy is caused by noisy data or by improper probe matching between platforms. We investigated whether the significant level of inconsistency between results produced by alternative gene expression microarray platforms could be reduced by stringent sequence matching of microarray probes. We mapped the short oligo probes of the Affymetrix platform onto cDNA clones of the Stanford microarray platform. Affymetrix probes were reassigned to redefined probe sets if they mapped to the same cDNA clone sequence, regardless of the original manufacturer-defined grouping. The NCI-60 gene expression profiles produced by Affymetrix HuFL platform were recalculated using these redefined probe sets and compared to previously published cDNA measurements of the same panel of RNA samples.     

Temporal and Spatial Analysis of Sin Nombre Virus Quasispecies in Naturally Infected Rodents

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (Peromyscus maniculatus), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3' small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.