Background: Members 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. Results: A 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. Conclusion: Our 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.

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.

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.

Frog 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.

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.

Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N⁶-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N⁶-isopentenyladenine (iP), N⁶-isopentenyladenine-9-riboside (iPR), N⁶-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N⁶-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.