Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.     

Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.     

Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase.

The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10(-3)) were observed at this (guanine) site. We sought evidence for RNA 3'-->5' exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[1-thio]triphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.     

Isolation of vesicular stomatitis virus defective interfering genomes with different amounts of 5''-terminal complementarity.

I isolated at least 30 different vesicular stomatitis virus defective interfering (DI) genomes, distinguished by chain length, by five independent undiluted passages of a repeatedly cloned virus plaque. Labeling of the 3' hydroxyl ends of these DI genomes and RNase digestion studies demonstrated that the ends of these DI genomes were terminally complementary to different extents (approximately 46 to 200 nucleotides). Mapping studies showed that the complementary ends of all of the DI genomes were derived from the 5' ends of the nondefective minus-strand genome. Regardless of the extent of terminal complementarity, all of the DI genomes synthesized the same 46-nucleotide minus-strand leader RNA.     

Mutation Frequencies at Defined Single Codon Sites in Vesicular Stomatitis Virus and Poliovirus Can Be Increased Only Slightly by Chemical Mutagenesis

Mutagenesis by a variety of chemical mutagens conferred only 1.1- to 2.8-fold increases in mutation frequencies at defined single base sites in vesicular stomatitis virus and poliovirus.     

Sequence Determination of the (+) Leader RNA Regions of the Vesicular Stomatitis Virus Chandipura, Cocal, and Piry Serotype Genomes

Using 3'-end-labeled genome probes, cells infected with vesicular stomatitis virus Chandipura, Cocal, and Piry serotypes were shown to contain (+) leader RNAs of approximately 50 nucleotides in length. The nucleotide sequence of the leader RNA regions of these genomes was determined and compared with the previously reported sequences of both the (+) and (-) leader RNA regions of other vesicular stomatitis virus serotypes. Regions of strong conservation of nucleotide sequence among the various vesicular stomatitis virus serotypes suggest those nucleotides thought to be involved in control functions during vesicular stomatitis virus replication.     

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.     

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.     

Continuing coevolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles

We quantitatively analyzed the interference interactions between defective interfering (DI) particles and mutants of cloned vesicular stomatitis virus passaged undiluted hundreds of times in BHK-21 cells. DI particles which predominated at different times in these serial passages always interfered most strongly (and very efficiently) with virus isolated a number of passages before the isolation of the DI particles. Virus isolated at the same passage level as the predominant DI particles usually exhibited severalfold resistance to these DI particles. Virus mutants (Sdi- mutants) isolated during subsequent passages always showed increasing resistance to these DI particles, followed by decreasing resistance as new DI particles arose to predominate and exert their own selective pressures on the virus mutant population. It appears that such coevolution of virus and DI particle populations proceeds indefinitely through multiple cycles of selection of virus mutants resistant to a certain DI particle (or DI particle class), followed by mutants resistant to a newly predominant DI particle, etc. At the peak of resistance, virus mutants were isolated which were essentially completely resistant to a particular DI particle; i.e., they were several hundred thousand-fold resistant, and they formed plaques of normal size and numbers in the presence of extremely high multiplicities of the DI particle. However, they were sensitive to interference by other DI particles. Recurring population interactions of this kind can promote rapid virus evolution. Complete sequencing of the N (nucleocapsid) and NS (polymerase associated) genes of numerous Sdi- mutants collected at passage intervals showed very few changes in the NS protein, but the N gene gradually accumulated a series of stable nucleotide and amino acid substitutions, some of which correlated with extensive changes in the Sdi- phenotype. Likewise, the 5' termini (and their complementary plus-strand 3' termini) continued to accumulate extensive base substitutions which were strikingly confined to the first 47 nucleotides. We also observed addition and deletion mutations in noncoding regions of the viral genome at a level suggesting that they probably occur at a high frequency throughout the genome, but usually with lethal or debilitating consequences when they occur in coding regions.     

Positive selection of synonymous mutations in vesicular stomatitis virus

Prevailing evolutionary forces are typically deduced from the pattern of differences in synonymous and non-synonymous mutations, under the assumption of neutrality in the absence of amino acid change. We determined the complete sequence of ten vesicular stomatitis virus populations evolving under positive selection. A significant number of the mutations occurred independently in two or more strains, a process known as parallel evolution, and a substantial fraction of the parallel mutations were silent. Parallel evolution was also identified in non-coding regions. These results indicate that silent mutations can significantly contribute to adaptation in RNA viruses, and relative frequencies of synonymous and non-synonymous substitutions may not be useful to resolve their evolutionary history.     

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.     

Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus

The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.     

Genome RNA terminus conservation and diversity among vesiculoviruses.

Sequence analysis of the RNA genome termini of various vesiculovirus standard and defective interfering (DI) particles demonstrated that some virus regulatory sequences and domains of virus N protein are highly conserved while others show considerable divergence. Clearly, distinct RNA signal sequences and protein-coding regions of these virus genomes have quite different evolutionary pressures or constraints. Terminal regions of DI-particle RNA genomes of these viruses were found to possess self-complementary stems at the RNA termini, demonstrating the conservation of this DI-particle structural feature throughout the vesiculovirus group. A high degree of conservation of the 3'-terminal sequences of recent and historic isolates of vesicular stomatitis virus New Jersey was also demonstrated.     

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.     

Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.