[show abstract][hide abstract] ABSTRACT: Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus) and puma (Puma concolor) blood cell DNA samples from California, Colorado and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4 kb region of each putative virus species including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology.Importance Gammaherpesviruses (GHVs) establish life-long infection in many animal species and can cause cancer and other diseases in humans and animals. In this study we identified DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus) and pumas (Puma concolor, also known as cougars or mountain lions). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified as native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the US. In contrast, puma virus was found only in a specific region of southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.
[show abstract][hide abstract] ABSTRACT: Alterations in the functional levels of cyclin-dependent kinase-8 (CDK8) or its partner, cyclin C, have been clearly associated with cancers, including colon cancer, melanoma, and osteosarcoma. Walleye dermal sarcoma virus encodes a retroviral cyclin (RV-cyclin) that localizes to interchromatin granule clusters and binds CDK8. It also binds to the Aα subunit (PR65) of protein phosphatase 2A (PP2A). Binding to the Aα subunit excludes the regulatory B subunit, but not the catalytic C subunit, in a manner similar to that of T antigens of the small DNA tumor viruses. The expression of the RV-cyclin enhances the activity of immune affinity-purified CDK8 in vitro for RNA polymerase II carboxy-terminal domain (CTD) and histone H3 substrates. PP2A also enhances CDK8 kinase activity in vitro for the CTD but not for histone H3. The PP2A enhancement of CDK8 is independent of RV-cyclin expression and likely plays a role in the normal regulation of CDK8. The manipulation of endogenous PP2A activity by inhibition, amendment, or depletion confirmed its role in CDK8 activation by triggering CDK8 autophosphorylation. Although RV-cyclin and PP2A both enhance CDK8 activity, their actions are uncoupled and additive in kinase reactions. PP2A may be recruited to CDK8 in the Mediator complex by a specific PP2A B subunit or additionally by the RV-cyclin in infected cells, but the RV-cyclin appears to activate CDK8 directly and in a manner independent of its physical association with PP2A.
Journal of Virology 02/2012; 86(10):5742-51. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus encodes a retroviral cyclin (rv-cyclin) with a cyclin box fold and transcription activation domain (AD). Co-immune precipitation (co-IP) identified an association of rv-cyclin with cyclin-dependent kinase 8 (cdk8). Cdk8 is dependent upon cyclin C and regulates transcription with the Mediator complex, a co-activator of transcription. Mutation of cyclin residues, required for cdk binding, disrupts rv-cyclin-cdk8 co-IP. Mutation or removal of the AD has no effect on cdk8 interaction. Direct rv-cyclin-cdk8 binding is demonstrated by pulldown of active cdk8 and by GST-rv-cyclin binding to recombinant cdk8. Cdk3 is also activated by cyclin C and phosphorylates retinoblastoma protein to initiate entry into the cell division cycle. Co-IP and pulldowns demonstrate direct rv-cyclin binding to cdk3 as well. The rv-cyclin functions as a structural ortholog of cyclin C in spite of its limited amino acid sequence identity with C cyclins or with any known cyclins.
[show abstract][hide abstract] ABSTRACT: Retroviruses have been detected in the majority of vertebrate species analyzed to date. In fish, thirteen proliferative diseases
have been associated with the presence of retroviruses. The etiologic relationship of retroviruses with these diseases is
primarily based on tumor-associated retrovirus-like particles, the presence of reverse transcriptase activity in neoplastic
tissues, and the ability to transmit disease with tumor extracts. Increased epizootics of cancers in fish, particularly in
farm-reared or hatchery facilities, have prompted the awareness and further study of the suspected retroviruses. Many proliferative
diseases in fish develop and regress seasonally and provide unique models for the study of cancer development and regression.
The complete proviral sequences of six fish retroviruses have revealed unique genome organizations and expression patterns.
Two simple retroviruses have been identified: an exogenous virus from Atlantic salmon swim-bladder tumors and an endogenous
retrovirus in the zebrafish genome. A complex retrovirus was isolated from a cultured snakehead-fish cell-line, although an
association with disease has not been shown. Three complex retroviruses isolated from walleye skin-proliferative diseases
display a similar genomic structure and encode three novel accessory proteins that contribute to oncogenesis and tumor regression.
The accessory proteins from walleye dermal-sarcoma virus (WDSV) function in the regulation of host and viral-gene expression
by altering cell-signaling pathways and induction of apoptosis. A new retroviral genus, Epsilonretroviruses, has been established based on the distinctive sequence and structure of the walleye viruses. Phylogenetic analyses show
a high degree of heterogeneity within the piscine retroviruses.
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish. Six stable transgenic lines expressing rv-cyclin from the constitutive CMVtk promoter were generated. Immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction demonstrate that rv-cyclin is widely expressed in different tissues in these fish. These lines were viable and histologically normal for up to 2 years. No increase in tumors or tissue proliferation was observed following N-ethyl N-nitrosourea exposure or following tail wounding and subsequent tissue regeneration compared to controls. These data indicate that rv-cyclin is not independently sufficient for tumor induction in zebrafish.
[show abstract][hide abstract] ABSTRACT: The retroviral cyclin protein (rv-cyclin) of walleye dermal sarcoma virus contains two known functional domains, a cyclin box motif and a carboxy terminal transcription activation domain (AD). The AD contacts TATA-binding protein-associated factor 9 (TAF9), and this action is necessary for both positive and negative regulation of transcription from host and viral promoters. Negative regulation occurs via interference with TAF9 binding by transcriptional activators. Transcription factors that share a functional TAF9-binding motif include NF-kappaB. Rv-cyclin down regulates NF-kappaB-dependent transcription, whether induced by TNFalpha or by direct phosphorylation of IkappaB by expressed MEKK1. In rv-cyclin-expressing cells, NF-kappaB p65 is phosphorylated and translocated to the nucleus, where it forms heterodimers with p50 and binds NF-kappaB response elements. Furthermore, interference with NF-kappaB is dependent upon an intact TAF9-binding motif in rv-cyclin. The outcome of this NF-kappaB down regulation is likely to be important in the control of virus replication and tumorigenesis.
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assays and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKCalpha, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. Virus expression is tightly regulated and limited to accessory gene transcripts throughout tumour development. During tumour regression, this regulation is lost and the replication of virus is greatly enhanced. Cultured walleye fibroblasts infected in vitro do not produce significant quantities of infectious virus. Tissue culture cells established by explantation of tumour cells were found to harbour WDSV provirus and to express accessory and structural proteins. The sequence of the provirus showed little variation from a previous WDSV isolate. Retroviral particles were isolated from supernatants from these cells and were able to transfer infection to uninfected walleye fibroblasts. In addition to the virus present in supernatants, much of the virus was cell associated and liberated only by sonication. This virus was found at internal cellular membranes, including mitochondria, and was infectious.
Journal of General Virology 10/2007; 88(Pt 9):2583-9. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. A WDSV accessory gene encodes a cyclin homolog or retroviral cyclin (rv-cyclin). WDSV rv-cyclin was found to be associated with transcription complexes and to affect transcription in a cell-type and promoter-dependent manner. It inhibited the WDSV promoter in walleye fibroblasts and activated transcription from GAL4 promoters when fused to the GAL4 DNA binding domain, and an activation domain (AD) has been localized to 30 amino acids in the carboxyl region. rv-cyclin can block the pulldown of transcription coactivators by the AD of VP16, and the isolated rv-cyclin AD interferes specifically with the interaction between the carboxyl halves of the VP16 AD, VP16C, and TATA-binding protein-associated factor 9 (TAF9). The carboxyl region and isolated AD can bind TAF9 directly in assays of protein-protein interaction in vitro. Furthermore, rv-cyclin and the isolated rv-cyclin AD interfere specifically with the function of VP16C in transcription assays. A previously identified motif within the VP16C sequence mediates TAF9 binding, and this motif is present in the activation domains of a variety of TAF9-binding transcriptional activators. A similar motif is present in the rv-cyclin AD, and point mutations within this motif affect rv-cyclin function and protein-protein interactions. The results support a model of transcription regulation by direct interaction with TAF9.
Journal of Virology 01/2007; 80(24):12041-8. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1(+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pull down is lost by mutation of the activation domain.
[show abstract][hide abstract] ABSTRACT: Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.
Journal of Virology 02/2005; 79(2):1125-32. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing tumors. To understand the role of host cell factors in WDSV expression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumor nuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.
Journal of Virology 08/2004; 78(14):7590-601. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcomas are associated with the presence of a complex retrovirus, walleye dermal sarcoma virus (WDSV). These sarcomas develop and regress seasonally in naturally infected fish. In addition to gag, pol and env, WDSV contains three open reading frames (ORFs), designated orf a, orf b and orf c. orf c is located between the 5' long terminal repeat and gag. Developing tumours contain low levels of orf a and orf b transcripts, whereas regressing tumours contain high levels of genomic transcripts and virus particles. Orf C protein is encoded by the full-length, genomic transcript and can be detected in tumour extracts with anti-Orf C-specific antisera. To determine the subcellular location of WDSV Orf C, cultured cells were transfected with an expression vector encoding haemagglutinin-tagged Orf C and examined by immunofluorescence. Orf C was observed throughout the cytoplasm and accumulated in cytoplasmic organelles. Dual-antibody staining for Orf C and mitochondrial cytochrome c demonstrated colocalization of Orf C with mitochondria and loss of the normal distribution of mitochondria in the cytoplasm. Cells transiently expressing Orf C exhibited apoptotic morphology and increased levels of surface phosphatidylserine and were unable to retain MitoTracker Orange, a dye that accumulates in active mitochondria. These results imply a functional role for WDSV Orf C in an alteration of mitochondrial function that results in apoptosis contributing to tumour regression.
Journal of General Virology 03/2003; 84(Pt 2):375-81. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.
Journal of Virology 09/2002; 76(16):8031-9. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Walleye dermal sarcoma virus (WDSV) induces tumors and allows or possibly directs tumor regression. WDSV encodes a putative cyclin homologue, Orf A, and six variant Orf A transcripts have been identified. Northern analysis indicated that a 3.3-kb transcript, encoding full-length Orf A, is the predominant transcript in developing, but not regressing, tumors. Three Orf A proteins, one full-length and two amino-truncated forms, were expressed in mammalian and piscine cells, and their intracellular locations were determined. The full-length form was nuclear and concentrated in interchromatin granule clusters, defined by colocalization with SC-35. The amino-truncated forms were cytoplasmic. Fusion of amino-terminal portions of Orf A to a heterologous protein demonstrated that residues 1-112 were necessary for nuclear localization. Mutation of aa K80 and/or E110 disrupted nuclear localization, suggesting a mechanism similar to that of cellular A- and D-type cyclins for its nuclear import.
[show abstract][hide abstract] ABSTRACT: Reverse transcriptase PCR (RT-PCR) consistently detected bovine leukemia virus transcripts in fresh cells, and competitive RT-PCR enumerated these transcripts. The detection of transcripts in limited numbers of tumor cells indicated that expression occurs in a minority of cells. The data suggest that individual cells contain hundreds of copies of the tax/rex transcript in vivo.
Journal of Virology 11/1999; 73(10):8890-7. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Green turtle fibropapillomatosis is a neoplastic disease of increasingly significant threat to the survivability of this species. Degenerate PCR primers that target highly conserved regions of genes encoding herpesvirus DNA polymerases were used to amplify a DNA sequence from fibropapillomas and fibromas from Hawaiian and Florida green turtles. All of the tumors tested (n= 23) were found to harbor viral DNA, whereas no viral DNA was detected in skin biopsies from tumor-negative turtles. The tissue distribution of the green turtle herpesvirus appears to be generally limited to tumors where viral DNA was found to accumulate at approximately two to five copies per cell and is occasionally detected, only by PCR, in some tissues normally associated with tumor development. In addition, herpesviral DNA was detected in fibropapillomas from two loggerhead and four olive ridley turtles. Nucleotide sequencing of a 483-bp fragment of the turtle herpesvirus DNA polymerase gene determined that the Florida green turtle and loggerhead turtle sequences are identical and differ from the Hawaiian green turtle sequence by five nucleotide changes, which results in two amino acid substitutions. The olive ridley sequence differs from the Florida and Hawaiian green turtle sequences by 15 and 16 nucleotide changes, respectively, resulting in four amino acid substitutions, three of which are unique to the olive ridley sequence. Our data suggest that these closely related turtle herpesviruses are intimately involved in the genesis of fibropapillomatosis.
[show abstract][hide abstract] ABSTRACT: Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.