On the expression strategy of the tospoviral genome

The work described in this thesis was aimed at the unravelling of the molecular biology of tospoviruses, with special emphasis on the process of replication of the tripartite RNA genome.At the onset of the research the complete genome sequence of tomato spotted wilt virus (TSWV), type species of the genus Tospovirus, became available. These sequence data indicated that the tospoviruses represent plant-infecting members of the large family of the arthropod-born Bunyaviridae. Genome sequence comparisons indicated however that the L RNA segment of TSWV would encode a much larger viral polymerase (331.5 kDa) than, as far as known, its animal-infecting counterparts (reported sizes of 241 to 259 kDa). To verify whether a large polymerase represents a characteristic i.e. genus-specific property of tospoviruses the complete sequence of the L RNA segment of a second tospovirus, impatiens necrotis spot virus (INSV), was elucidated (Chapter 2). These sequence data revealed that the L RNA of INSV appeared to be comparable in size to that of TSWV (8675 nucleotides versus 8897 for TSWV), containing an open reading frame with a predicted size of 330.3 kDa of the INSV polymerase. Therefore the next question to be answered was whether the large primary translation product of the tospoviral L RNA acts as an unprocessed polymerase or whether this protein would undergo some cleavages to obtain smaller, functional replication proteins. Answering this question was even more necessary since the theoretical size of the TSWV L RNA ORF greatly exceeded previously determined sizes (110 to 220 kDa) for a large protein reported to copurify with TSWV particles. To this end both the 5'-terminal and 3'-terminal parts of the ORF in the TSWV L RNA were expressed in Escherichia coli and antibodies raised against these regions. Using these tools it could be established that the polymerase (L protein) of TSWV, though significantly larger than that of other bunyaviruses, is present in virus particles (10 to 20 copies per virion) in an unprocessed, full length form (Chapter 3). To allow further analyses of the TSWV polymerase, attempts were made to clone and express the complete L RNA ORF in the baculovirus/insect cell system. In spite of all efforts, only a shorter translation product of 67 kDa was obtained from a baculovirus recombinant containing a complete DNA copy of the TSWV L RNA (Chapter 3). Sequence analysis of the cloned copy revealed a 80 basepairs deletion, resulting in two premature stop codons, which most likely have led to the resulting truncated L protein.To gain more insight in the "cap-snatching" event which takes place during initiation of tospovirus transcription, nucleoprotein (N) mRNAs were partially purified from TSWV-infected N.rustica leaves and cloned (Chapter 4). Sequence analysis of the cloned, 5'-proximate regions of 20 cloned mRNAs showed the presence of extra, non-templated sequences, ranging in length from 12 to 21 nucleotides, confirming our earlier primer extension studies. As these sequences were of non-viral origin a cap-snatching mechanism for tospoviral transcription initiation could thus be definitively identified. None of the hostderived leader sequences analyzed were identical and only limited sequence specificity at the endonucleolytic site was observed (some preference for cleavage at a U residue). During the course of this Ph.D. research, Adkins et al. (1995) reported that in vitro transcriptase activity was associated with freshly isolated TSWV particles. It was investigated (Chapter 5) whether the reported levels of in vitro activity could be further improved and whether this system would lend itself for analysis of the viral proteins involved by e.g. inhibition studies using specific antibodies. Trichloroacetic acid-precipitable products could consistently be obtained after incubation of detergent-disrupted TSWV virions under the assay conditions reported by Adkins et al. (1995) and using (α- 32P)CTP. No significant improvement in CMP incorporation levels could be achieved by testing variable conditions and varying concentrations of assay components. The reaction products obtained hybridized with clones from all three genomic RNA segments. No discrimination between transcription and replication could be made however, and since none of the available specific antibodies directed against any viral protein had an inhibitory effect, it was concluded that the current in vitro system will be of limited value for unravelling the RNA synthesizing process and the role of the individual viral proteins therein.As a first step towards a manipulatable transcription/replication system, a hybrid baculovirus/bacteriophage T7 vector system was developed for transient expression in insect cells of all factors involved in TSWV genome transcription and replication. The results obtained (Chapter 6) illustrate the potential of the system. Although various foreign genes could successfully be expressed to measurable amounts, the reconstitution of a TSWV transcription/replication complex was hampered due to the apparent impossibility (Chapter 3) to clone the complete polymerase gene. Finally, in Chapter 7 (General discussion and concluding remarks), the results obtained are compared with the data reported for animalinfecting bunyaviruses, leading to a discussion of some evolutionary aspects. Furthermore, suggestions are made to circumvent some of the problems encountered during the course of the studies presented in this thesis

Satisfiability, sequence niches, and molecular codes in cellular signaling

Biological information processing as implemented by regulatory and signaling networks in living cells requires sufficient specificity of molecular interaction to distinguish signals from one another, but much of regulation and signaling involves somewhat fuzzy and promiscuous recognition of molecular sequences and structures, which can leave systems vulnerable to crosstalk. This paper examines a simple computational model of protein-protein interactions which reveals both a sharp onset of crosstalk and a fragmentation of the neutral network of viable solutions as more proteins compete for regions of sequence space, revealing intrinsic limits to reliable signaling in the face of promiscuity. These results suggest connections to both phase transitions in constraint satisfaction problems and coding theory bounds on the size of communication codes

Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication

Exploring the Complexity of the HIV-1 Fitness Landscape

Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1. We find that these landscapes are characterized by large correlation lengths, considerable neutrality, and high ruggedness and that these properties depend only weakly on whether fitness is measured in the absence or presence of different antiretrovirals. Accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. While the relative extent to which mutations affect fitness on their own (main effects) or in combination with other mutations (epistasis) is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged, less neutral, and more correlated than expected from the distribution of main effects and epistatic interactions alone. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place

How Protein Stability and New Functions Trade Off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (ΔΔG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (ΔΔG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied ΔΔG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to ΔΔG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average ΔΔG = +0.9 kcal/mol), and are almost as destabilizing as the “average” mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently “silent” mutations in regions of the protein that are irrelevant to its function

Initial Mutations Direct Alternative Pathways of Protein Evolution

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations

Neutrality and Robustness in Evo-Devo: Emergence of Lateral Inhibition

Embryonic development is defined by the hierarchical dynamical process that translates genetic information (genotype) into a spatial gene expression pattern (phenotype) providing the positional information for the correct unfolding of the organism. The nature and evolutionary implications of genotype–phenotype mapping still remain key topics in evolutionary developmental biology (evo-devo). We have explored here issues of neutrality, robustness, and diversity in evo-devo by means of a simple model of gene regulatory networks. The small size of the system allowed an exhaustive analysis of the entire fitness landscape and the extent of its neutrality. This analysis shows that evolution leads to a class of robust genetic networks with an expression pattern characteristic of lateral inhibition. This class is a repertoire of distinct implementations of this key developmental process, the diversity of which provides valuable clues about its underlying causal principles