Negative effects of chemical mutagenesis on the adaptive behavior of vesicular stomatitis virus.

Changes in adaptability of vesicular stomatitis virus (VSV) upon treatment with chemical mutagens have been investigated. Results showed no improvement in virus viability or adaptability at any given level of mutagenesis. In fact, increasing inhibition of virus production and adaptability was observed with increasing levels of mutagenesis. This was true for all tested VSV variants replicating either in changing or constant host cell environments. Results also showed that mutagen-treated RNA virus populations which had undergone severe fitness declines were able to recover lost fitness completely after several large-population passages in BHK21, cells. The present findings illustrate the highly optimized states of RNA viruses and their potential to adapt readily. These results are significant for the possible development of specific antiviral agents designed to be mutagenic.     

Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.     

Host alternation is necessary to maintain the genome stability of rift valley fever virus

Background

Most arthropod-borne viruses (arboviruses) are RNA viruses, which are maintained in nature by replication cycles that alternate between arthropod and vertebrate hosts. Arboviruses appear to experience lower rates of evolution than RNA viruses that replicate in a single host. This genetic stability is assumed to result from a fitness trade-off imposed by host alternation, which constrains arbovirus genome evolution. To test this hypothesis, we used Rift Valley fever virus (RVFV), an arbovirus that can be transmitted either directly (between vertebrates during the manipulation of infected tissues, and between mosquitoes by vertical transmission) or indirectly (from one vertebrate to another by mosquito-borne transmission).

Methodology/Principal Findings

RVFV was serially passaged in BHK21 (hamster) or Aag2 (Aedes aegypti) cells, or in alternation between the two cell types. After 30 passages, these single host-passaged viruses lost their virulence and induced protective effects against a challenge with a virulent virus. Large deletions in the NSs gene that encodes the virulence factor were detectable from the 15^th serial passage onwards in BHK21 cells and from the 10^th passage in Aag2 cells. The phosphoprotein NSs is not essential to viral replication allowing clones carrying deletions in NSs to predominate as they replicate slightly more rapidly. No genetic changes were found in viruses that were passaged alternately between arthropod and vertebrate cells. Furthermore, alternating passaged viruses presenting complete NSs gene remained virulent after 30 passages.

Conclusions/Significance

Our results strongly support the view that alternating replication is necessary to maintain the virulence factor carried by the NSs phosphoprotein.     

Large-population passages of vesicular stomatitis virus in interferon-treated cells select variants of only limited resistance.

Vesicular stomatitis virus (VSV) populations were repeatedly passaged in L-929 cells treated with alpha interferon (IFN-alpha) at levels of 25 U/ml. This IFN-alpha concentration induced a 99.9% inhibition of viral yield in standard infections. Analysis of viral fitness (overall replicative ability measured in direct competition with a reference wild-type VSV) after 21 passages in IFN-treated cells showed only a limited increase or no increase in fitness, compared with the greater increase upon parallel passage in cells not treated with IFN-alpha. However, this limited increase in fitness was more pronounced when competition assays were carried out with IFN-alpha-treated cells, suggesting the selection of VSV populations with a low level of resistance to IFN-alpha. Thus, despite the extensively documented capacity of VSV to adapt to changing environments, the antiviral state induced by IFN-alpha imposes adaptive constraints on VSV which are not readily overcome.     

Effect of alternating passage on adaptation of sindbis virus to vertebrate and invertebrate cells

Mosquito-borne alphaviruses, which replicate alternately and obligately in mosquitoes and vertebrates, appear to experience lower rates of evolution than do many RNA viruses that replicate solely in vertebrates. This genetic stability is hypothesized to result from the alternating host cycle, which constrains evolution by imposing compromise fitness solutions in each host. To test this hypothesis, Sindbis virus was passaged serially, either in one cell type to eliminate host alteration or alternately between vertebrate (BHK) and mosquito (C6/36) cells. Following 20 to 50 serial passages, mutations were identified and changes in fitness were assessed using competition assays against genetically marked, surrogate parent viruses. Specialized viruses passaged in a single cell exhibited more mutations and amino acid changes per passage than those passaged alternately. Single host-adapted viruses exhibited fitness gains in the cells in which they specialized but fitness losses in the bypassed cell type. Most but not all viruses passaged alternately experienced lesser fitness gains than specialized viruses, with fewer mutations per passage. Clonal populations derived from alternately passaged viruses also exhibited adaptation to both cell lines, indicating that polymorphic populations are not required for simultaneous fitness gains in vertebrate and mosquito cells. Nearly all passaged viruses acquired Arg or Lys substitutions in the E2 envelope glycoprotein, but enhanced binding was only detected for BHK cells. These results support the hypothesis that arbovirus evolution may be constrained by alternating host transmission cycles, but they indicate a surprising ability for simultaneous adaptation to highly divergent cell types by combinations of mutations in single genomes.     

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.     

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.     

Cost of host radiation in an RNA virus

Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host. However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats.     

An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.     

Temperature-Sensitive Virus from Aedes albopictus Cells Chronically Infected with Sindbis Virus

Cultures of Aedes albopictus cells persistently infected with wild-type Sindbis virus (SV-W) give rise to small plaque-forming mutants which are also temperature sensitive. These mutants, designated SV-C, are neutralized by antiserum produced against SV-W. Mutant ts clones were isolated from SV-C by plaque purification. After serial undiluted passage in BHK or mosquito cells, each of the clones gave rise to ts(+) revertants which, however, remained mutant with respect to plaque morphology. Nineteen of 20 clones derived from SV-C were RNA(+), and one was RNA(-) (SV-C-2). The RNA synthesizing activity, once induced in infected cells by SV-C-2, was stable at the nonpermissive temperature (39.5 C). All clones derived from SV-C were inactivated at 60 C much more quickly than was SV-W. It was not possible to demonstrate complementation between any of the SV-C clones.     

Contingent Neutrality in Competing Viral Populations

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

Evolutionary genomics of host adaptation in vesicular stomatitis virus

Populations experiencing similar selection pressures can sometimes diverge in the genetic architectures underlying evolved complex traits. We used RNA virus populations of large size and high mutation rate to study the impact of historical environment on genome evolution, thus increasing our ability to detect repeatable patterns in the evolution of genetic architecture. Experimental vesicular stomatitis virus populations were evolved on HeLa cells, on MDCK cells, or on alternating hosts. Turner and Elena (2000. Cost of host radiation in an RNA virus. Genetics. 156:1465-1470.) previously showed that virus populations evolved in single-host environments achieved high fitness on their selected hosts but failed to increase in fitness relative to their ancestor on the unselected host and that alternating-host-evolved populations had high fitness on both hosts. Here we determined the complete consensus sequence for each evolved population after 95 generations to gauge whether the parallel phenotypic changes were associated with parallel genomic changes. We also analyzed the patterns of allele substitutions to discern whether differences in fitness across hosts arose through true pleiotropy or the presence of not only a mutation that is beneficial in both hosts but also 1 or more mutations at other loci that are costly in the unselected environment (mutation accumulation [MA]). We found that ecological history may influence to what extent pleiotropy and MA contribute to fitness asymmetries across environments. We discuss the degree to which current genetic architecture is expected to constrain future evolution of complex traits, such as host use by RNA viruses.     

Extreme heterogeneity in populations of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) sequence evolution and population heterogeneity were examined by T1 oligonucleotide mapping. Individual clones isolated from clonal pools of wild-type Indiana serotype VSV displayed identical T1 maps. This was observed even after one passage at high concentrations of the potent viral mutagen 5-fluorouracil. Under low-multiplicity passage conditions, the consensus T1 fingerprint of this virus remained unchanged after 523 passages. Interestingly, however, individual clones from this population (passage 523) differed significantly from each other and from consensus sequence. When virus population equilibria were disrupted by high-multiplicity passage (in which defective interfering particle interference is maximized) or passage in the presence of mutagenic levels of 5-fluorouracil, rapid consensus sequence evolution occurred and extreme population heterogeneity was observed (with some members of these population differing from others at hundreds of genome positions). A limited sampling of clones at one stage during high-multiplicity passages suggested the presence of at least several distinct master sequences, the related subpopulations of which exhibit at least transient competitive fitness within the total virus population (M. Eigen and C.K. Biebricher, p. 211-245, in E. Domingo, J.J. Holland, P. Ahlquist, ed., RNA Genetics, vol. 3, 1988). These studies further demonstrate the important role of selective pressure in determining the genetic composition of RNA virus populations. This is true under equilibrium conditions in which little consensus sequence evolution is observed owing to stabilizing selection as well as under conditions in which selective pressure is driving rapid RNA virus genome evolution.     

Subclonal components of consensus fitness in an RNA virus clone.

Most RNA virus populations exhibit extremely high mutation frequencies which generate complex, genetically heterogeneous populations referred to as quasi-species. Previous work has shown that when a large spectrum of the quasi-species is transferred, natural selection operates, leading to elimination of noncompetitive (inferior) genomes and rapid gains in fitness. However, whenever the population is repeatedly reduced to a single virion, variable declines in fitness occur as predicted by the Muller's ratchet hypothesis. Here, we quantitated the fitness of 98 subclones isolated from an RNA virus clonal population. We found a normal distribution around a lower fitness, with the average subclone being less fit than the parental clonal population. This finding demonstrates the phenotypic diversity in RNA virus populations and shows that, as expected, a large fraction of mutations generated during virus replication is deleterious. This clarifies the operation of Muller's ratchet and illustrates why a large number of virions must be transferred for rapid fitness gains to occur. We also found that repeated genetic bottleneck passages can cause irregular stochastic declines in fitness, emphasizing again the phenotypic heterogeneity present in RNA virus populations. Finally, we found that following only 60 h of selection (15 passages in which virus yields were harvested after 4 h), RNA virus populations can undergo a 250% average increase in fitness, even on a host cell type to which they were already well adapted. This is a remarkable ability; in population biology, even a much lower fitness gain (e.g., 1 to 2%) can represent a highly significant reproductive advantage. We discuss the biological implications of these findings for the natural transmission and pathogenesis of RNA viruses.     

Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity

The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 DeltaK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 DeltaK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K-->F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.