Craig R. Brunetti

Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes.

BackgroundMembers of the family Iridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of the Iridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members.ResultsA series of genomic sequence comparisons were made among, and between the Ranavirus and Megalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, the Megalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26.ConclusionOur re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.

Frog virus 3-like infections in aquatic amphibian communities.

Frog virus 3 (FV3) and FV3-like viruses, are members of the genus Ranavirus (family Iridoviridae), and they have been associated with infectious diseases that may be contributing to amphibian population declines. We examined the mode of transmission of an FV3-like virus, and potential hosts and reservoirs of the virus in a local amphibian community. Using the polymerase chain reaction to detect infected animals, we found an FV3-like virus in south-central Ontario, Canada, amphibian communities, where it infects sympatric amphibian species, including ranid and hylid tadpoles (Rana sylvatica, Hyla versicolor, and Pseudacris spp.), larval salamanders (Ambystoma spp.), and adult eastern-spotted newts (Notophthalmus viridescens). The high prevalence of FV3-like infections in caudate larvae suggests that salamanders are likely to be both hosts and reservoirs. In laboratory FV3 challenges of R. sylvatica, the rate of infection was dependent on the amount of virus to which the animals were exposed. In addition, although vertical transmission was suspected, horizontal transmission through exposure to infected pond water is the most likely route of infection in tadpoles. Based on our observations, a simple model of FV3/FV3-like virus transmission postulates that, in aquatic amphibian communities, transmission of the virus occurs between anuran and urodele species, with ambystomatid salamanders the most likely reservoir for the ranavirus in our study.

Analysis of an orf virus chemokine-binding protein: Shifting ligand specificities among a family of poxvirus viroceptors

We identify a secreted chemokine inhibitor encoded by orf virus (ORFV), the prototypic poxvirus of the Parapoxvirus genus, and show that it is related to the poxvirus type II CC-chemokine-binding proteins (CBP-II) produced by members of the Orthopoxvirus and Leporipoxvirus genera. The ORFV chemokine-binding protein (CBP) is functionally similar to the CBP-II proteins in its ability to bind and inhibit many CC-chemokines with high affinity. However, unlike CBP-II, the ORFV CBP also binds with high affinity to lymphotactin, a member of the C-chemokine family, demonstrating that the ORFV CBP possesses an altered binding specificity. Interestingly, the amino acid sequence of ORFV CBP more closely resembles the granulocyte–macrophage colony-stimulating factor/IL-2 inhibitory factor also produced by ORFV, implicating the granulocyte–macrophage colony-stimulating factor/IL-2 inhibitory factor protein as a highly diverged, but related, member of the CBP-II protein family. Notably, these findings suggest that the genes that encode these proteins derive from a common poxvirus ancestral gene that has since been modified in binding specificity during speciation of the poxvirus genera. Overall, these findings illustrate the concept of evolution of viral proteins at the biophysical and molecular interface.

Complete Genomic Sequence and Comparative Analysis of the Tumorigenic Poxvirus Yaba Monkey Tumor Virus

ABSTRACT 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.

The Genomic Diversity and Phylogenetic Relationship in the Family Iridoviridae

The Iridoviridae family are large viruses (∼120–200 nm) that contain a linear double-stranded DNA genome. The genomic size of Iridoviridae family members range from 105,903 bases encoding 97 open reading frames (ORFs) for frog virus 3 to 212,482 bases encoding 211 ORFs for Chilo iridescent virus. The family Iridoviridae is currently subdivided into five genera: Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus. Iridoviruses have been found to infect invertebrates and poikilothermic vertebrates, including amphibians, reptiles, and fish. With such a diverse array of hosts, there is great diversity in gene content between different genera. To understand the origin of iridoviruses, we explored the phylogenetic relationship between individual iridoviruses and defined the core-set of genes shared by all members of the family. In order to further explore the evolutionary relationship between the Iridoviridae family repetitive sequences were identified and compared. Each genome was found to contain a set of unique repetitive sequences that could be used in future virus identification. Repeats common to more than one virus were also identified and changes in copy number between these repeats may provide a simple method to differentiate between very closely related virus strains. The results of this paper will be useful in identifying new iridoviruses and determining their relationship to other members of the family.

SIMPLE/LITAF Expression Induces the Translocation of the Ubiquitin Ligase Itch towards the Lysosomal Compartments

LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.

Complete genome analysis of a frog virus 3 (FV3) isolate and sequence comparison with isolates of differing levels of virulence

BackgroundFrog virus 3 (FV3) is the type species of the genus Ranavirus, and in the past few decades, FV3 infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia. The reasons for the pathogenicity of FV3 are not well understood.FindingsWe investigated three FV3 isolates designated SSME, wt-FV3, and aza-Cr, and reported that our wt-FV3 and aza-Cr strains showed similar levels of virulence, while SSME was the least virulent in an in vivo study with Lithiobates pipiens tadpoles. Using 454 GS-FLX sequencing technology, we sequenced SSME and compared it to the published wt-FV3 genome. SSME had multiple amino acid deletions in ORFs 49/50L, 65L, 66L, and 87L, which may explain its reduced virulence. We also investigated repeat regions and found that repeat copy number differed between isolates, with only one group of 3 isolates and 1 pair of isolates being identical at all 3 locations.ConclusionsIn this study we have shown that genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select repeat locations. These genomic areas with deletions/insertions may represent regions that affect virulence, and therefore require investigation. Furthermore, we have identified repeat regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions.

Yaba Monkey Tumor Virus Encodes a Functional Inhibitor of Interleukin-18

ABSTRACT Interleukin-18 (IL-18) is a critical proinflammatory cytokine whose extracellular bioactivity is regulated by a cellular IL-18 binding protein (IL-18BP). Many poxviruses have acquired variants of this IL-18BP gene, some of which have been shown to act as viral virulence factors. Yaba monkey tumor virus (YMTV) encodes a related family member, 14L, which is similar to the orthopoxvirus IL-18BPs. YMTV 14L was expressed from a baculovirus system and tested for its ability to bind and inhibit IL-18. We found that YMTV 14L bound both human IL-18 (hIL-18) and murine IL-18 with high affinity, at 4.1 nM and 6.5 nM, respectively. YMTV 14L was able to fully sequester hIL-18 but could only partially inhibit the biological activity of hIL-18 as measured by gamma interferon secretion from KG-1 cells. Additionally, 17 hIL-18 point mutants were tested by surface plasmon resonance for their ability to bind to YMTV 14L. Two clusters of hIL-18 surface residues were found to be important for the hIL-18-YMTV 14L interaction, in contrast to results for the Variola virus IL-18BP, which has been shown to primarily interact with a single cluster of three amino acids. The altered binding specificity of YMTV 14L most likely represents an adaptation resulting in increased fitness of the virus and affirms the plasticity of poxviral inhibitor domains that target cytokines like IL-18.

Cellular LITAF interacts with frog virus 3 75L protein and alters its subcellular localization.

ABSTRACT Iridoviruses are a family of large double-stranded DNA (dsDNA) viruses that are composed of 5 genera, including the Lymphocystivirus, Ranavirus, Megalocytivirus, Iridovirus, and Chloriridovirus genera. The frog virus 3 (FV3) 75L gene is a nonessential gene that is highly conserved throughout the members of the Ranavirus genus but is not found in other iridoviruses. FV3 75L shows high sequence similarity to a conserved domain found in the C terminus of LITAF, a small cellular protein with unknown function. Here we show that FV3 75L localizes to early endosomes, while LITAF localizes to late endosomes/lysosomes. Interestingly, when FV3 75L and LITAF are cotransfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosomes/lysosomes, where FV3 75L then colocalizes with LITAF. In addition, we demonstrated that virally produced 75L colocalizes with LITAF. We confirmed a physical interaction between LITAF and FV3 75L but found that this interaction was not mediated by two PPXY motifs in the N terminus of LITAF. Mutation of two PPXY motifs in LITAF did not affect the colocalization of LITAF and FV3 75L but did change the location of the two proteins from late endosomes/lysosomes to early endosomes.

LITAF Mutations Associated with Charcot-Marie-Tooth Disease 1C Show Mislocalization from the Late Endosome/Lysosome to the Mitochondria

Charcot-Marie-Tooth (CMT) disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T). The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.