Reovirus genomic diversity confers plasticity for protease utility during adaptation to intracellular uncoating.

IMPORTANCE: Reoviruses infect many mammals and are widely studied as a model system for enteric viruses. However, most of our reovirus knowledge comes from laboratory strains maintained on immortalized L929 cells. Herein, we asked whether naturally circulating reoviruses possess the same genetic and phenotypic characteristics as laboratory strains. Naturally circulating reoviruses obtained from sewage were extremely diverse genetically. Moreover, sewage reoviruses exhibited poor fitness on L929 cells and relied heavily on gut proteases for viral uncoating and productive infection compared to laboratory strains. We then examined how naturally circulating reoviruses might adapt to cell culture conditions. Within three passages, virus isolates from the parental sewage population were selected, displaying improved fitness and intracellular uncoating in L929 cells. Remarkably, selected progeny clones were present at 0.01% of the parental population. Altogether, using reovirus as a model, our study demonstrates how the high genetic diversity of naturally circulating viruses results in rapid adaptation to new environments.

p38 Mitogen-Activated Protein Kinase Signaling Enhances Reovirus Replication by Facilitating Efficient Virus Entry, Capsid Uncoating, and Postuncoating Steps.

Mammalian orthoreovirus serotype 3 Dearing is an oncolytic virus currently undergoing multiple clinical trials as a potential cancer therapy. Previous clinical trials have emphasized the importance of prescreening patients for prognostic markers to improve therapeutic success. However, only generic cancer markers such as epidermal growth factor receptor (EGFR), Hras, Kras, Nras, Braf, and p53 are currently utilized, with limited benefit in predicting therapeutic efficacy. This study aimed to investigate the role of p38 mitogen-activated protein kinase (MAPK) signaling during reovirus infection. Using a panel of specific p38 MAPK inhibitors and an inactive inhibitor analogue, p38 MAPK signaling was found to be essential for establishment of reovirus infection by enhancing reovirus endocytosis, facilitating efficient reovirus uncoating at the endo-lysosomal stage, and augmenting postuncoating replication steps. Using a broad panel of human breast cancer cell lines, susceptibility to reovirus infection corresponded with virus binding and uncoating efficiency, which strongly correlated with status of the p38ß isoform. Together, results suggest p38ß isoform as a potential prognostic marker for early stages of reovirus infection that are crucial to successful reovirus infection. IMPORTANCE The use of Pelareorep (mammalian orthoreovirus) as a therapy for metastatic breast cancer has shown promising results in recent clinical trials. However, the selection of prognostic markers to stratify patients has had limited success due to the fact that these markers are upstream receptors and signaling pathways that are present in a high percentage of cancers. This study demonstrates that the mechanism of action of p38 MAPK signaling plays a key role in establishment of reovirus infection at both early entry and late replication steps. Using a panel of breast cancer cell lines, we found that the expression levels of the MAPK11 (p38ß) isoform are a strong determinant of reovirus uncoating and infection establishment. Our findings suggest that selecting prognostic markers that target key steps in reovirus replication may improve patient stratification during oncolytic reovirus therapy.

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly.

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins µ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Kinetic analysis of virus +RNA production, core versus OC protein expression, and core particles versus whole virus particle accumulation, indicated that assembly of OC proteins onto core particles was temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a µ1-dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC µ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating µ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.

Closely related reovirus lab strains induce opposite expression of RIG-I/IFN-dependent versus -independent host genes, via mechanisms of slow replication versus polymorphisms in dsRNA binding σ3 respectively.

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.

Breast Tumor-Associated Metalloproteases Restrict Reovirus Oncolysis by Cleaving the σ1 Cell Attachment Protein and Can Be Overcome by Mutation of σ1.

Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyomavirus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein σ1, which diminished virus binding to sialic acid (SA)-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated σ1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231, and HS578T breast cancer cell lines recapitulated σ1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers and that breast cancer cells by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of SA levels on a panel of cancer cells showed that truncated σ1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in σ1 that prevents σ1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, toxicity equivalent to that of wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data show that tumor microenvironments have the potential to reduce infectivity of reovirus.IMPORTANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have a negative impact on proteinaceous cancer therapies, such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System.

Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions.

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid µ1 protein to µ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

Vaccinia virus Western Reserve induces rapid surface expression of a host molecule detected by the antibody 4C7 that is distinct from CLEC2D.

In this study, the effect of active infection with vaccinia virus Western Reserve (VACV WR) on expression of C-type lectin domain family 2 (CLEC2D), a ligand of the human NK cell inhibitory receptor NKR-P1, was examined. As predicted, VACV infection led to a loss of CLEC2D mRNA in 221 cells, a B cell lymphoma line. Surprisingly, VACV infection of 221 cells caused a dramatic increase in cell surface staining for one CLEC2D-specific antibody, 4C7. There were no changes in other antibodies specific for CLEC2D and no indication that NK cells with NKR-P1A were inhibited, suggesting 4C7 detects a non-CLEC2D molecule following infection. The rapid increase in 4C7 signal requires virus attachment and is disrupted by UV treatment, but does not depend on new transcription or translation of either cellular or viral proteins. 4C7 does react with intracellular compartments, suggesting the molecule that is detected at the surface following infection is derived from an intracellular store. The phenomenon extends beyond lymphoid cells: it was observed in the non-human primate cell line Cos-7, but not with myxoma, a poxvirus distinct from VACV. To our knowledge, this is the first report of VACV or any poxvirus leading to rapid externalization of a host molecule. Among the VACV strains tested, the phenomenon was restricted to VACV WR and IHD-W, suggesting it has a virulence-, as opposed to a replication-related, function.

Elimination of mitochondrial DNA is not required for herpes simplex virus 1 replication.

UNLABELLED: Infection with herpes simplex virus type 1 (HSV-1) results in the rapid elimination of mitochondrial DNA (mtDNA) from host cells. It is known that a mitochondrial isoform of the viral alkaline nuclease (UL12) called UL12.5 triggers this process. However, very little is known about the impact of mtDNA depletion on viral replication or the biology of HSV-1 infections. These questions have been difficult to address because UL12.5 and UL12 are encoded by overlapping transcripts that share the same open reading frame. As a result, mutations that alter UL12.5 also affect UL12, and UL12 null mutations severely impair viral growth by interfering with the intranuclear processing of progeny viral genomes. Therefore, to specifically assess the impact of mtDNA depletion on viral replication, it is necessary to eliminate the activity of UL12.5 while preserving the nuclear functions of UL12. Previous work has shown that the human cytomegalovirus alkaline nuclease UL98 can functionally substitute for UL12 during HSV-1 replication. We found that UL98 is unable to deplete mtDNA in transfected cells and therefore generated an HSV-1 variant in which UL98 coding sequences replace the UL12/UL12.5 open reading frame. The resulting virus was severely impaired in its ability to trigger mtDNA loss but reached titers comparable to those of wild-type HSV-1 in one-step and multistep growth experiments. Together, these observations demonstrate that the elimination of mtDNA is not required for HSV-1 replication in cell culture. IMPORTANCE: Herpes simplex virus types 1 and 2 destroy the DNA of host cell mitochondria, the powerhouses of cells. Epstein-Barr virus, a distantly related herpesvirus, has a similar effect, indicating that mitochondrial DNA destruction is under positive selection and thus confers a benefit to the virus. The present work shows that mitochondrial DNA destruction is not required for efficient replication of herpes simplex virus type 1 in cultured Vero kidney epithelial cells, suggesting that this activity likely benefits the virus in other cell types or in the intact human host.

Herpes simplex virus protein kinases US3 and UL13 modulate VP11/12 phosphorylation, virion packaging, and phosphatidylinositol 3-kinase/Akt signaling activity.

UNLABELLED: The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays key roles in diverse cellular activities and promotes cell growth and survival. It is therefore unsurprising that most viruses modify this pathway in order to facilitate their replication and spread. Previous work has suggested that the herpes simplex virus 1 (HSV-1) tegument proteins VP11/12 and US3 protein kinase modulate the PI3K/Akt pathway, albeit in opposing ways: VP11/12 binds and activates Src family kinases (SFKs), is tyrosine phosphorylated, recruits PI3K in an SFK-dependent fashion, and is required for HSV-induced phosphorylation of Akt on its activating residues; in contrast, US3 inhibits Akt activation and directly phosphorylates downstream Akt targets. We examined if US3 negatively regulates Akt by dampening the signaling activity of VP11/12. Consistent with this hypothesis, the enhanced Akt activation that occurs during US3-null infection requires VP11/12 and correlates with an increase in SFK-dependent VP11/12 tyrosine phosphorylation. In addition, deleting US3 leads to a striking increase in the relative abundances of several VP11/12 species that migrate with reduced mobility during SDS-PAGE. These forms arise through phosphorylation, strictly require the viral UL13 protein kinase, and are excluded from virions. Taken in combination, these data indicate that US3 dampens SFK-dependent tyrosine and UL13-dependent serine/threonine phosphorylation of VP11/12, thereby inhibiting VP11/12 signaling and promoting virion packaging of VP11/12. These results illustrate that protein phosphorylation events mediated by viral protein kinases serve to coordinate the roles of VP11/12 as a virion component and intracellular signaling molecule. IMPORTANCE: Herpesvirus tegument proteins play dual roles during the viral life cycle, serving both as structural components of the virus particle and as modulators of cellular and viral functions in infected cells. How these two roles are coordinated during infection and virion assembly is a fundamental and largely unanswered question. Here we addressed this issue with herpes simplex virus VP11/12, a tegument protein that activates the cellular PI3K/Akt signaling pathway. We showed that protein phosphorylation mediated by the viral US3 and UL13 kinases serves to orchestrate its functions: UL13 appears to inhibit VP11/12 virion packaging, while US3 antagonizes UL13 action and independently dampens VP11/12 signaling activity.

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

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.

Frog virus 3 open reading frame 97R localizes to the endoplasmic reticulum and induces nuclear invaginations.

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.

Antibody dependent enhancement of frog virus 3 infection.

BACKGROUND: Viruses included in the family Iridoviridae are large, icosahedral, dsDNA viruses that are subdivided into 5 genera. Frog virus 3 (FV3) is the type species of the genus Ranavirus and the best studied iridovirus at the molecular level. Typically, antibodies directed against a virus act to neutralize the virus and limit infection. Antibody dependent enhancement occurs when viral antibodies enhance infectivity of the virus rather than neutralize it. RESULTS: Here we show that anti-FV3 serum present at the time of FV3 infection enhances infectivity of the virus in two non-immune teleost cell lines. We found that antibody dependent enhancement of FV3 was dependent on the Fc portion of anti-FV3 antibodies but not related to complement. Furthermore, the presence of anti-FV3 serum during an FV3 infection in a non-immune mammalian cell line resulted in neutralization of the virus. Our results suggest that a cell surface receptor specific to teleost cell lines is responsible for the enhancement. CONCLUSIONS: This report represents the first evidence of antibody dependent enhancement in iridoviruses. The data suggests that anti-FV3 serum can either neutralize or enhance viral infection and that enhancement is related to a novel antibody dependent enhancement pathway found in teleosts that is Fc dependent.

Expression of frog virus 3 genes is impaired in mammalian cell lines.

Frog virus 3 (FV3) is a large DNA virus that is the prototypic member of the family Iridoviridae. To examine levels of FV3 gene expression we generated a polyclonal antibody against the FV3 protein 75L. Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection. While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection. One explanation for the lack of gene expression in mammalian cell lines may be inefficient codon usage. As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells. Therefore, although FV3 can infect and replicate in mammalian cell lines, the virus may not express its full complement of genes due to inefficient codon usage in mammalian species.

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

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.

Accumulation of endogenous LITAF in aggresomes.

LITAF is a 161 amino acid cellular protein which includes a proline rich N-terminus and a conserved C-terminal domain known as the simple-like domain. Mutations in LITAF have been identified in Charcot-Marie tooth disease, a disease characterized by protein aggregates. Cells transfected with cellular LITAF reveal that LITAF is localized to late endosomes/lysosomes. Here we investigated the intracellular localization of endogenous LITAF. We demonstrated that endogenous LITAF accumulates at a discrete cytoplasmic site in BGMK cells that we identify as the aggresome. To determine the domain within LITAF that is responsible for the localization of LITAF to aggresomes, we created a construct that contained the C-terminal simple-like domain of LITAF and found that this construct also localizes to aggresomes. These data suggest the simple-like domain is responsible for targeting endogenous LITAF to the aggresome.

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.

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.

Characterization of the promoter activity of a poxvirus conserved element.

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.