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. 1999 Jun;73(6):5123-31.
doi: 10.1128/JVI.73.6.5123-5131.1999.

Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys

B Damania  1 , M Li, J K Choi, L Alexander, J U Jung, R C Desrosiers
Affiliations

Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys

B Damania et al. J Virol. 1999 Jun.

Abstract

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi's sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.

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Figures

FIG. 1
FIG. 1
Analysis of ORFs present in the K29 clone. Any ORF larger than 50 amino acids is indicated by stippling. Complete ORFs for CCPH, DHFR, and R1 are depicted, along with the partial ORF of the mDBP gene (encoding the first 109 amino acids of the mDBP) (asterisk). The arrows indicate the direction of expression for each gene.
FIG. 2
FIG. 2
Genomic organization of the left end of the RRV, KSHV, and HVS genomes. The first ORFs, R1, K1, and STP, are depicted. The DHFR gene constitutes the second ORF in RRV and HVS but is displaced further downstream in KSHV. The third and fourth ORFs in the RRV and HSV genomes are the CCPH4 and mDBP6 genes. Because of the displacement of DHFR in KSHV, CCPH and mDBP6 are the second and third ORFs in KSHV. U1, U2, and U5 indicate the locations of coding sequences for small U-RNAs (18, 20). The arrows indicate the direction of gene expression.
FIG. 3
FIG. 3
Organization of the structural regions of the R1 and K1 proteins. N represents the amino termini of the two proteins, and the hatched boxes represent their putative transmembrane domains. The N-terminal extracellular domains have approximately the same number of amino acids (aa) and contain cysteines that may form disulfide linkages similar to other members of the immunoglobulin superfamily. The cytoplasmic tail of K1 contains one ITAM in which a YXXL and YXXP are separated by 7 amino acids (16, 17). The cytoplasmic tail of R1 contains five potential YXXL SH2-binding motifs separated by 3 to 9 amino acids.
FIG. 4
FIG. 4
Amino acid identity and similarity between the R1 and K1 proteins. The N-terminal 245 amino acids of R1 were compared to the equivalent region of K1. These two proteins exhibit 27% identity and 40% similarity in their extracellular domains. +, amino acid similarity; −, gaps in amino acid sequence. The dotted lines represent the signal peptide sequence.
FIG. 5
FIG. 5
Detection of the R1 protein. The R1 gene was tagged with an AU1 epitope and expressed in Cos-1 cells. Lysates were run on SDS-PAGE under reducing conditions (lanes 1 to 5) or nonreducing conditions (lane 6) and transferred to nitrocellulose. Western blotting was performed with an anti-AU1 antibody. Lanes: 1, lysates of cells transfected with the control vector; 2, lysates of Cos-1 cells transfected with the R1 expression vector; 3, membrane fractions from Cos-1 cells transfected with R1; 4, lysates of R1-transfected Cos-1 cells treated with tunicamycin; 5, membrane fractions of R1-transfected Cos-1 cells treated with tunicamycin; 6, lysate of R1-transfected Cos-1 cells (nonreducing condition of SDS-PAGE).
FIG. 6
FIG. 6
Localization of R1 protein. Bosc-23 (top panels) or Cos-1 (bottom panels) cells were transfected with the expression vector alone or a vector expressing a C-terminal AU1-tagged R1 protein (R1 CAU1). At 48 h posttransfection, immunofluorescence was performed with an anti-AU1 antibody. The R1 protein was localized to the cytoplasmic and plasma membranes.
FIG. 7
FIG. 7
Amino acid sequence of the R1 gene from four different macaque rhadinovirus isolates. H-Mm26-95 is the original M. mulatta isolate. H-Mm309-95 was also isolated from M. mulatta (a different animal). H-Mf23-97 was isolated from M. fascicularis, and H-Mn19545 was isolated from M. nemestrina. The dotted lines represent the signal peptide sequence, the box represents the transmembrane domain, and the five putative SH2-binding motifs are underlined in bold. The R1 protein shows a high degree of homology to the immunoglobulin receptor superfamily in the N-terminal 220 amino acids.
FIG. 8
FIG. 8
Phylogenetic analysis of R1 sequences. An evolutionary tree based on the amino acid sequences encoded by the four R1 genes from four macaque rhadinovirus isolates (H-Mm26-95, H-Mm309-95, H-Mf23-97, and H-Mn19545) is shown. The phenogram was obtained by the distance method, using the neighbor-joining program. The R1 sequences cluster together and are more closely related to each other than to K1.
FIG. 9
FIG. 9
Transforming activity of the R1 protein. (A) Puromycin-resistant cells obtained after transfection with a pBabe control retroviral vector (top left) or a pBabe-STP (top right) or pBabe-R1 (bottom)-expressing vector were plated at 106 cells per 100-mm tissue culture dish. After 14 days of incubation, the cells were photographed to show morphologic changes. (B) The cells were stained with methylene blue to show foci of transformed cells.
FIG. 9
FIG. 9
Transforming activity of the R1 protein. (A) Puromycin-resistant cells obtained after transfection with a pBabe control retroviral vector (top left) or a pBabe-STP (top right) or pBabe-R1 (bottom)-expressing vector were plated at 106 cells per 100-mm tissue culture dish. After 14 days of incubation, the cells were photographed to show morphologic changes. (B) The cells were stained with methylene blue to show foci of transformed cells.
FIG. 10
FIG. 10
Construction of recombinant HVSΔSTP/R1. The diagram shows the strategy used to make the recombinant HVSΔSTP/R1. The detailed procedure has been described previously (9).
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References

    1. Boshoff C, Whitby D, Hatziannou T, Fisher C, Van der Walt J, Hatzakis A, Weiss R A, Schultz T F. Kaposi’s sarcoma associated herpesvirus in HIV-negative Kaposi sarcoma. Lancet. 1995;345:1043–1044. - PubMed
    1. Burkhardt A L, Bolen J B, Kieff E, Longnecker R. An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases. J Virol. 1992;66:5161–5167. - PMC - PubMed
    1. Cambier J C. Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM) J Immunol. 1995;155:3281–3285. - PubMed
    1. Cesarman E, Nador R, Knowles D M. Body cavity based lymphoma in an HIV-seronegative patient without Kaposi’s sarcoma associated herpesvirus-like DNA sequences. N Engl J Med. 1996;334:273. - PubMed
    1. Cesarman E, Chang Y, Moore P S, Said J W, Knowles D M. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med. 1995;332:1186–1191. - PubMed

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