Relationships between polydnavirus genomes and viral gene expression

Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication.     

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.     

Finding candidate genes under positive selection in Non‐model species: examples of genes involved in host specialization in pathogens

Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host‐pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17 000–32 000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty‐two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non‐model organisms for which candidate genes for diversifying selection are needed.     

Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis

Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+) channels. To determine if these viral K(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+) channel pore modules from seven phycodnaviruses to the K(+) channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+) channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+) channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+) channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+) channels in algae and perhaps even all cellular organisms.     

Positive selection in the evolution of cancer

We hypothesize that forms of antagonistic coevolution have forged strong links between positive selection at the molecular level and increased cancer risk. By this hypothesis, evolutionary conflict between males and females, mothers and foetuses, hosts and parasites, and other parties with divergent fitness interests has led to rapid evolution of genetic systems involved in control over fertilization and cellular resources. The genes involved in such systems promote cancer risk as a secondary effect of their roles in antagonistic coevolution, which generates evolutionary disequilibrium and maladaptation. Evidence from two sources: (1) studies on specific genes, including SPANX cancer/testis antigen genes, several Y-linked genes, the pem homebox gene, centromeric histone genes, the breast cancer gene BRCA1, the angiogenesis gene ANG, cadherin genes, cytochrome P450 genes, and viral oncogenes; and (2) large-scale database studies of selection on different functional categories of genes, supports our hypothesis. These results have important implications for understanding the evolutionary underpinnings of cancer and the dynamics of antagonistically-coevolving molecular systems.     

Positive selection signatures in the TLR7 family

While adaptive immunity genes evolve rapidly under the influence of positive selection, innate immune system genes are known to evolve slowly due to strong purifying selection. Among the sensors of the innate immune system, Toll-like receptors (TLRs) are particularly important due to their ability to recognize and respond to pathogen-associated molecular patterns (PAMP), such as lipopolysaccharides, peptidoglycans, and nucleic acids from bacteria or viruses. In the present study, we examine the evolutionary process that has operated on the TLR7 family genes TLR7, TLR8, and TLR9. The results demonstrate that the average Ka/Ks (the ratio between nonsynonymous and synonymous substitution rates) of each TLR family gene is far lower than one regardless of estimating methods, supporting previous observations of strong purifying selection in this gene family. Interestingly, however, analysis of Ka/Ks ratios along the coding regions of TLR7 family genes by sliding-window analysis reveals a few narrow high peaks (Ka/Ks > 1). The most prominent peak corresponds to a specific region in the ectodomain, which exists only in the TLR7 family, suggesting that this unique structure of the TLR7 family might have been a target of positive selection in a variety of lineages. Furthermore, maximum likelihood model tests suggest that positive selection is the best explanation for a certain fraction of the amino acid substitutions in the TLR9.     

Patterns of evolutionary selection pressure in the immune signaling protein TRAF3IP2 in mammals

TRAF3 interacting protein 2 (TRAF3IP2) is important for immune responses to pathogens, inflammatory signals and autoimmunity in mammals. In the present study, we collected 19 mammalian TRAF3IP2 sequences and investigated the various types of selection pressure acting on them. Maximum likelihood estimations of nonsynonymous (dN) to synonymous (dS) substitution (dN/dS) ratios for the aligned coding sequences indicated that, as a whole, TRAF3IP2 has been subject to purifying selection. However, the N-terminus of the protein has been subject to higher selection pressure than the C-terminal domain. While eight amino acid residues within the N-terminus appear to have evolved under positive selection, no evidence for such selection was found in the C-terminus. The positively selected residues, which fall outside the currently known functional sites within TRAF3IP2, may have novel functions. The different selection pressures acting on the N- and C-terminal regions are consistent with their protein structures: the C-terminal structure is an ordered structure, whereas the N-terminus is disordered. Taken together with the results of previous studies, it is plausible that positive selection on the N-terminus of TRAF3IP2 may have occurred by competitive coevolution between mammalian hosts and viruses.     

Anti-apoptotic genes of baculoviruses

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.     

Diversifying and Purifying Selection in the Peptide Binding Region of <Emphasis Type="Italic">DRB</Emphasis> in Mammals

The class II genes of the major histocompatibility complex encode proteins which play a crucial role in antigen presentation. They are among the most polymorphic proteins known, and this polymorphism is thought to be the result of natural selection. To understand the selective pressure acting on the protein and to examine possible differences in the evolutionary dynamics among species, we apply maximum likelihood models of codon substitution to analyze the DRB genes of six mammalian species: human, chimpanzee, macaque, tamarin, dog, and cow. The models account for variable selective pressures across codons in the gene and have the power to detect amino acid residues under either positive or negative selection. Our analysis detected positive selection in the DRB genes in each of the six mammals examined. Comparison with structural data reveals that almost all amino acid residues inferred to be under positive selection in humans are in the peptide binding region (PBR) and are in contact with the antigen side chains, although residues outside of but close to the PBR are also detected. Strong purifying selection is also detected in the PBR, at sites which contact the antigen and at sites which may be involved in dimerization or T cell binding. The analysis demonstrates the utility of the random-sites analysis even when structural information is available. The different mammalian species are found to share many positively or negatively selected sites, suggesting that their functional roles have remained very similar in the different species, despite the different habitats and pathogens of the species.     

Viral biogeography revealed by signatures in Sulfolobus islandicus genomes

Viruses are a driving force of microbial evolution. Despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. One of the primary factors that define viral population structure is coevolution with microbial hosts. Experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. The biogeographic distribution of viruses that infect Sulfolobus islandicus is investigated using genome comparisons among four newly identified, integrated, Sulfolobus spindle-shaped viruses and previously sequenced viral strains. Core gene sequences show a biogeographic distribution where viral genomes are specifically associated with each local population. In addition, signatures of host–virus interactions recorded in the sequence-specific CRISPR (clustered regularly interspaced short palindromic repeats) system show that hosts have interacted with viral communities that are more closely related to local viral strains than to foreign ones. Together, both proviral and CRISPR sequences show a clear biogeographic structure for Sulfolobus viral populations. Our findings demonstrate that virus–microbe coevolution must be examined in a spatially explicit framework. The combination of host and virus biogeography suggests a model for viral diversification driven by host immunity and local adaptation.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.     

Solution structure of the carboxyl-terminal cysteine-rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.

Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.     

MHC polymorphism under host-pathogen coevolution

The genes encoding major histocompatibility (MHC) molecules are among the most polymorphic genes known for vertebrates. Since MHC molecules play an important role in the induction of immune responses, the evolution of MHC polymorphism is often explained in terms of increased protection of hosts against pathogens. Two selective pressures that are thought to be involved are (1) selection favoring MHC heterozygous hosts, and (2) selection for rare MHC alleles by host-pathogen coevolution. We have developed a computer simulation of coevolving hosts and pathogens to study the relative impact of these two mechanisms on the evolution of MHC polymorphism. We found that heterozygote advantage per se is insufficient to explain the high degree of polymorphism at the MHC, even in very large host populations. Host-pathogen coevolution, on the other hand, can easily account for realistic polymorphisms of more than 50 alleles per MHC locus. Since evolving pathogens mainly evade presentation by the most common MHC alleles in the host population, they provide a selective pressure for a large variety of rare MHC alleles. Provided that the host population is sufficiently large, a large set of MHC alleles can persist over many host generations under host-pathogen coevolution, despite the fact that allele frequencies continuously change.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Suppressors of RNAi from plant viruses are subject to episodic positive selection

Viral suppressors of RNAi (VSRs) are proteins that actively inhibit the antiviral RNA interference (RNAi) immune response, providing an immune evasion route for viruses. It has been hypothesized that VSRs are engaged in a molecular ‘arms race’ with RNAi pathway genes. Two lines of evidence support this. First, VSRs from plant viruses display high sequence diversity, and are frequently gained and lost over evolutionary time scales. Second, Drosophila antiviral RNAi genes show high rates of adaptive evolution. Here, we investigate whether VSRs diversify faster than other genes and, if so, whether this is a result of positive selection, as might be expected in an arms race. By analysis of 12 plant RNA viruses, we show that the relative rate of protein evolution is higher for VSRs than for other genes, but that this is not attributable to pervasive positive selection. We argue that, because evolutionary time scales are extremely different for viruses and eukaryotes, it is improbable that viral adaptation (as measured by the ratio of non-synonymous to synonymous change) will be dominated by one-to-one coevolution with eukaryotes. Instead, for plant virus VSRs, we find strong evidence of episodic selection—diversifying selection that acts on a subset of lineages—which might be attributable to frequent shifts between different host genotypes or species.     

Two-step infection processes can lead to coevolution between functionally independent infection and resistance pathways

There is growing evidence that successful infection of hosts by pathogens requires a series of independent steps. However, how multistep infection processes affect host-pathogen coevolution is unclear. We present a coevolutionary model, inspired by empirical observations from a range of host-pathogen systems, where the infection process consists of the following two steps: the first is for the pathogen to recognize and locate a suitable host, and the second is to exploit the host while evading immunity. Importantly, these two steps conform to different models of infection genetics: inverse-gene-for-gene (IGFG) and gene-for-gene (GFG), respectively. We show that coevolution under this scenario can lead to coupled gene frequency changes across these two systems. In particular, selection often favors pathogens that are infective at the first, IGFG, step and hosts that are resistant at the second, GFG, step. Hence, there may be signals of positive selection between functionally independent systems whenever there are multistep processes determining resistance and infectivity. Such multistep infection processes are a fundamental, but overlooked feature of many host-pathogen interactions, and have important consequences for our understanding of host-pathogen coevolution.     

A geographical mosaic of coevolution in a slave-making host-parasite system

Three different isolated populations of the slave‐making ant Rossomyrmex minuchae, sympatric with its obligate host Proformica longiseta, are known from the high mountains of southern Spain. To test the prediction that the slave‐maker and its host represent a coevolutionary geographical mosaic, we studied the variation in the cuticular hydrocarbons (CHCs) as the trait most likely to show the selection mosaic, plus trait remixing by the gene flow in the populations of each species by means of microsatellites. We found within populations, host and parasite had more similar CHC profiles than between the populations or between parasites and allopatric hosts. The differences between the CHC profiles of the host and parasite, which may be responsible for the level of tolerance towards the parasite, varied between the populations suggesting the existence of a selection mosaic of coevolution. Furthermore, P. longiseta showed higher gene flow than R. minuchae, which would allow local variation in the coevolution of the host and parasite while allowing some trait remixing.     

Molecular dissection of <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedrovirus <Emphasis Type="Italic">orf8</Emphasis> gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac16 interacts with baculoviral and cellular proteins. Bm8/Ac16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bm8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.