Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome

Increasingly complex bioinformatic analysis is necessitated by the plethora of sequence information currently available. A total of 21 poxvirus genomes have now been completely sequenced and annotated, and many more genomes will be available in the next few years. First, we describe the creation of a database of continuously corrected and updated genome sequences and an easy-to-use and extremely powerful suite of software tools for the analysis of genomes, genes, and proteins. These tools are available free to all researchers and, in most cases, alleviate the need for using multiple Internet sites for analysis. Further, we describe the use of these programs to identify conserved families of genes (poxvirus orthologous clusters) and have named the software suite POCs, which is available at www.poxvirus.org. Using POCs, we have identified a set of 49 absolutely conserved gene families-those which are conserved between the highly diverged families of insect-infecting entomopoxviruses and vertebrate-infecting chordopoxviruses. An additional set of 41 gene families conserved in chordopoxviruses was also identified. Thus, 90 genes are completely conserved in chordopoxviruses and comprise the minimum essential genome, and these will make excellent drug, antibody, vaccine, and detection targets. Finally, we describe the use of these tools to identify necessary annotation and sequencing updates in poxvirus genomes. For example, using POCs, we identified 19 genes that were widely conserved in poxviruses but missing from the vaccinia virus strain Tian Tan 1998 GenBank file. We have reannotated and resequenced fragments of this genome and verified that these genes are conserved in Tian Tan. The results for poxvirus genes and genomes are discussed in light of evolutionary processes.     

Genome reduction by deletion of paralogs in the marine cyanobacterium Prochlorococcus

Several isolates of the marine cyanobacterial genus Prochlorococcus have smaller genome sizes than those of the closely related genus Synechococcus. In order to test whether loss of protein-coding genes has contributed to genome size reduction in Prochlorococcus, we reconstructed events of gene family evolution over a strongly supported phylogeny of 12 Prochlorococcus genomes and 9 Synechococcus genomes. Significantly, more events both of loss of paralogs within gene families and of loss of entire gene families occurred in Prochlorococcus than in Synechococcus. The number of nonancestral gene families in genomes of both genera was positively correlated with the extent of genomic islands (GIs), consistent with the hypothesis that horizontal gene transfer (HGT) is associated with GIs. However, even when only isolates with comparable extents of GIs were compared, significantly more events of gene family loss and of paralog loss were seen in Prochlorococcus than in Synechococcus, implying that HGT is not the primary reason for the genome size difference between the two genera.     

A selectable and excisable marker system for the rapid creation of recombinant poxviruses

Background

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.

Methodology/Principal Findings

Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.

Conclusion/Significance

The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.     

Technical knockout: understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes

The study of viral pathogens with genomes as large and complex as poxviruses represents a constant experimental challenge. Advances in recombinant DNA technologies have provided sophisticated methods to produce mutants defective in one or more viral genes, termed knockout (KO) viruses, thereby facilitating research into the impact of specific gene products on viral pathogenesis. Such strategies have rapidly advanced the systematic mining of many poxvirus genomes and enabled researchers to identify and characterize poxvirus genes whose functions represent the culmination of host and pathogen coevolution. Of particular interest are the multiple classes of virus-encoded immunomodulatory proteins that have evolved specifically to allow poxviruses to evade, obstruct or subvert critical elements within the host innate and acquired immune responses. Functional characterization of these viral genes by generating KO viruses and investigating the phenotypic changes that result is an important tool for understanding the molecular mechanisms underlying poxvirus replication and pathogenesis. Moreover, the insights gained have led to new developments in basic and clinical virology, provided a basis for the design of new vaccines and antivirals, and increased the potential application of poxviruses as investigative tools and sources of biotherapeutics for the treatment of human diseases.     

The Genome of Swinepox Virus

Swinepox virus (SWPV), the sole member of the Suipoxvirus genus of the Poxviridae, is the etiologic agent of a worldwide disease specific for swine. Here we report the genomic sequence of SWPV. The 146-kbp SWPV genome consists of a central coding region bounded by identical 3.7-kbp inverted terminal repeats and contains 150 putative genes. Comparison of SWPV with chordopoxviruses reveals 146 conserved genes encoding proteins involved in basic replicative functions, viral virulence, host range, and immune evasion. Notably, these include genes with similarity to genes for gamma interferon (IFN-gamma) receptor, IFN resistance protein, interleukin-18 binding protein, IFN-alpha/beta binding protein, extracellular enveloped virus host range protein, dUTPase, hydroxysteroid dehydrogenase, superoxide dismutase, serpin, herpesvirus major histocompatibility complex inhibitor, ectromelia virus macrophage host range protein, myxoma virus M011L, variola virus B22R, four ankyrin repeat proteins, three kelch-like proteins, five vaccinia virus (VV) A52R-like family proteins, and two G protein-coupled receptors. The most conserved genomic region is centrally located and corresponds to the VV region located between genes F9L and A38L. Within the terminal 13 kbp, colinearity is disrupted and multiple poxvirus gene homologues are absent or share a lower percentage of amino acid identity. Most of these differences involve genes and gene families with likely functions involving viral virulence and host range. Three open reading frames (SPV018, SPV019. and SPV020) are unique for SWPV. Phylogenetic analysis, genome organization, and amino acid identity indicate that SWPV is most closely related to the capripoxvirus lumpy skin disease virus, followed by the yatapoxvirus yaba-like disease virus and the leporipoxviruses. The gene complement of SWPV better defines Suipoxvirus within the Chordopoxvirinae subfamily and provides a basis for future genetic comparisons.     

The genomes of sheeppox and goatpox viruses

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae, are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.     

Horizontal gene transfer accelerates genome innovation and evolution

Horizontal gene transfer (HGT) spreads genetic diversity by moving genes across species boundaries. By rapidly introducing newly evolved genes into existing genomes, HGT circumvents the slow step of ab initio gene creation and accelerates genome innovation. However, HGT can only affect organisms that readily exchange genes (exchange communities). In order to define exchange communities and understand the internal and external environmental factors that regulate HGT, we analyzed approximately 20,000 genes contained in eight free-living prokaryotic genomes. These analyses indicate that HGT occurs among organisms that share similar factors. The most significant are genome size, genome G/C composition, carbon utilization, and oxygen tolerance.     

What tangled web: barriers to rampant horizontal gene transfer

Dawkins in his The Selfish Gene(1) quite aptly applies the term "selfish" to parasitic repetitive DNA sequences endemic to eukaryotic genomes, especially vertebrates. Doolittle and Sapienza(2) as well as Orgel and Crick(3) enlivened this notion of selfish DNA with the identification of such repetitive sequences as remnants of mobile elements such as transposons. In addition, Orgel and Crick(3) associated parasitic DNA with a potential to outgrow their host genomes by propagating both vertically via conventional genome replication as well as infectiously by horizontal gene transfer (HGT) to other genomes. Still later, Doolittle(4) speculated that unchecked HGT between unrelated genomes so complicates phylogeny that the conventional representation of a tree of life would have to be replaced by a thicket or a web of life.(4) In contrast, considerable data now show that reconstructions based on whole genome sequences are consistent with the conventional "tree of life".(5-10) Here, we identify natural barriers that protect modern genome populations from the inroads of rampant HGT.     

Evolutionary genomics of mycovirus-related dsRNA viruses reveals cross-family horizontal gene transfer and evolution of diverse viral lineages

ABSTRACT: BACKGROUND: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. RESULTS: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. CONCLUSIONS: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.     

Complete genomic sequence and comparative analysis of the tumorigenic poxvirus Yaba monkey tumor virus

The Yatapoxvirus genus of poxviruses is comprised of Yaba monkey tumor virus (YMTV), Tanapox virus, and Yaba-like disease virus (YLDV), which all have the ability to infect primates, including humans. Unlike other poxviruses, YMTV induces formation of focalized histiocytomas upon infection. To gain a greater understanding of the Yatapoxvirus genus and the unique tumor formation properties of YMTV, we sequenced the 134,721-bp genome of YMTV. The genome of YMTV encodes at least 140 open reading frames, all of which are also found as orthologs in the closely related YLDV. However, 13 open reading frames found in YLDV are completely absent from YMTV. Common to both YLDV and YMTV are the unusually large noncoding regions between many open reading frames. To determine whether any of these noncoding regions might be functionally significant, we carried out a comparative analysis between the putative noncoding regions of YMTV and similar noncoding regions from other poxviruses. This approach identified three new gene poxvirus families, defined as orthologs of YMTV23.5L, YMTV28.5L, and YMTV120.5L, which are highly conserved in virtually all poxvirus species. Furthermore, the comparative analysis also revealed a 40-bp nucleotide sequence at approximately 14,700 bases from the left terminus that was 100% identical in the comparable intergene site within members of the Yatapoxvirus, Suipoxvirus, and Capripoxvirus genera and 95% conserved in the Leporipoxvirus genus. This conserved sequence was shown to function as a poxvirus late promoter element in transfected and infected cells, but other functions, such as an involvement in viral replication or packaging, cannot be excluded. Finally, we summarize the predicted immunomodulatory protein repertoire in the Yatapoxvirus genus as a whole.     

Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses

Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.     

Evidence of horizontal gene transfer between human and animal commensal Escherichia coli strains identified by microarray

Bacteria exchange genetic material by horizontal gene transfer (HGT). To evaluate the impact of HGT on Escherichia coli genome plasticity, 19 commensal strains collected from the intestinal floras of humans and animals were analyzed by microarrays. Strains were hybridized against an oligoarray containing 2700 E. coli K12 chromosomal genes. A core (genes shared among compared genomes) and a flexible gene pool (genes unique for each genome) have been identified. Analysis of hybridization signals evidenced 1015 divergent genes among the 19 strains and each strain showed a specific genomic variability pattern. Four hundred and fifty-eight genes were characterized by higher rates of interstrain variation and were considered hyperdivergent. These genes are not randomly distributed onto the chromosome but are clustered in precise regions. Hyperdivergent genes belong to the flexible gene pool and show a specific GC content, differing from that of the chromosome, indicating acquisition by HGT. Among these genes, those involved in defense mechanisms and cell motility as well as intracellular trafficking and secretion were far more represented than others. The observed genome plasticity contributes to the maintenance of genetic diversity and may therefore be a source of evolutionary adaptation and survival.     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses.

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.