Jay Doniger

Human herpesvirus 6 (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53

The 357 amino acid open reading frame 1 (ORF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashanchi et al., Virology, 201, 95u2009–u2009106, 1994). In the current study, the SalI-L transforming region was localized to the SalI-L-SH subfragment. Several ORFs identified in SalI-L-SH by sequence analysis were cloned into a selectable mammalian expression vector, pBK-CMV. Only pBK/ORF1 transformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein produced fibrosarcomas when injected into nude mice, whereas control cells, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic. Western blot analysis of proteins extracted from the tumors revealed ORF-1 protein. Additional studies indicated that ORF-1 was expressed in HHV-6-infected human T-cells by 18u2009h. Co-immunoprecipitation experiments showed that ORF-1 protein bound to tumor suppressor protein p53, and the ORF-1 binding domain on p53 was located between residues 28 and 187 of p53, overlapping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in truncated ORF-1, expressing cells. Importantly, the truncated ORF-1 mutant also failed to cause transformation. Analysis of several human tumors by PCR revealed ORF-1 DNA sequences in some angioimmunoblastic lymphadenopathies, Hodgkins and non-Hodgkins lymphomas and glioblastomas. The detection of ORF-1 sequences in human tumors, while not proof per se, is a prerequisite for establishing its role in tumor development. Taken together, the results demonstrate that ORF-1 is an HHV-6 oncogene that binds to and affects p53. The identification of both transforming and transactivating activities within ORF-1 is a characteristic of other viral oncogenes and is the first reported for HHV-6.

Characterization of the human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) oncogene, Kaposin (ORF K12)

BACKGROUNDnHuman herpesvirus 8 (HHV-8) has been implicated in the etiology of Kaposis sarcoma (KS), a highly angiogenic tumor of complex histology, and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castlemans disease (MCD). A number of HHV-8 encoded genes have been proposed to be involved in the pathogenesis of KS and PEL and a few have been shown to be oncogenic in heterologous systems (Reyes GR, LaFemina R, Hayward SD, Hayward GS. Morphological transformation by DNA fragments of human herpesviruses: evidence for two distinct transforming regions in herpes simplex virus types 1 and 2 and lack of correlation with biochemical transfer of the thymidine kinase gene. Cold Spring Harbor Symp Quant Biol 1980;44:629-641; Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 1996;274:1739-1744; Cheng EH, Nicholas J, Bellows DS, Hayward GS, Guo HG, Reitz MS, Hardwick JM. A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak. Proc Natl Acad Sci USA 1997;94:690-694; Li M, Lee H, Yoon DW, Albrecht JC, Fleckenstein B, Neipel F, Jung JU. Kaposis sarcoma-associated herpesvirus encodes a functional cyclin. J Virol 1997;71:1984-1991; Neipel F, Albrecht J-C, Fleckenstein B. Cell-homologous genes In the Kaposis sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J Virol 1997;71:4187-4192; Nicholas J, Ruvolo VR, Burns WH, Sandford G, Wan X, Ciufo D, Hendrickson SB, Guo HG, Hayward GS, Reitz MS. Kaposis sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6. Nat Med 1997;3:287-292; Nicholas J, Zong J, Alcendor DJ, Ciufu DM, Poole LJ, Sarisky RT, Chiuo C, Zhang X, Wan X, Guo H, Reitz MS, Hayward GS. Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV-8. JNCI Monographs 1998;23:79-88; Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposis sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The kaposin gene (ORF K12) encoded by the abundant latency-associated HHV-8 transcript, T0.7, has been previously shown to induce tumorigenic transformation of Rat-3 cells (Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposis sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The current study is a further characterization of kaposin protein.nnnOBJECTIVESnCharacterization of kaposin expression in transformed and tumor-derived Rat-3 cells as well as PEL cell lines, BCBL-1, BC-3 and KS-1 and analysis of mechanism(s) of transformation.nnnDESIGNnThe presence of kaposin DNA in transformed cells was determined by fluorescent in situ hybridization (FISH). Expression of kaposin protein was analyzed by Western blot analysis and indirect immunofluorescence assay (IFA). (ABSTRACT TRUNCATED)

Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains

The unr gene was identified as a transcription unit located immediately upstream of N-ras in the genome of several mammalian species. While this genetic organization could be important for the transcriptional regulation of unr and N-ras, the function of the protein product of unr is unknown. unr is ubiquitously expressed and codes for an 85 kDa protein which is not closely related to previously characterized proteins. Nevertheless, a search for protein motifs has indicated the presence of five cold shock domains within unr, a motif present in procaryotic cold shock proteins and in the vertebrate Y box factors. As these proteins have been reported to interact with nucleic acids, we investigated whether unr could bind to some classes of nucleic acids. We report here that unr has a high affinity for single-stranded DNA or RNA and a low affinity for double-stranded nucleic acids. Its low affinity for double-stranded DNA clearly distinguishes unr from the Y box factors. The binding of unr to RNA does not appear to depend upon extended sequence motifs but requires some level of sequence complexity as unr has only a low affinity for most simple polymers including polyA stretches. unr is also characterized by its low affinity for double-stranded and structured RNAs. We further determined that unr is mostly localized in the cytoplasm, and is in part associated with the endoplasmic reticulum. These studies indicate that unr is a novel single-stranded nucleic acid binding protein which is likely to be associated with cytoplasmic mRNA in vivo.

A 79 amino acid oncogene is responsible for human cytomegalovirus mtrII induced malignant transformation

SummaryHuman cytomegalovirus (HCMV) morphological transforming region (mtr)II is the only HCMV mtr that was retained and expressed in transformed mouse or rat cells. The minimal transforming region has previously been shown to be within a 980-bpBanII/XhoI subfragment which encodes three open reading frames (ORF) of 34, 79, and 83 amino acids. This report provides definitive evidence that the 79-aa ORF is responsible for mtrII mediated tumorigenic transformation. The 79-aa ORF, subcloned into a mammalian expression vector, pCHC79orf, induced morphologic transformation of NIH 3T3 cells. These transformed cells expressed 79-aa ORF specific transcripts and were tumorigenic when injected into nude mice. A construct containing a triple termination linker inserted after codon 24 failed to transform NIH 3T3 cells to tumorigenicity even though 79-aa ORF specific transcripts were expressed. Furthermore, when the triple termination linker was inserted after codon 49, tumorigenic transformation still occurred. These results demonstrate that the 79-aa ORF is the oncogene within HCMV mtrII and that the first 49-aa are sufficient.

Identification of Two Promoters within Human Cytomegalovirus Morphologic Transforming Region II

A 980-bp subfragment of human cytomegalovirus (HCMV) strain Towne has been previously identified as morphologic transforming region II (mtrII) because of its ability to induce focal transformation of NIH 3T3 cells. Transcripts from this region, which could encode the three open reading frames (ORFs), 79, 83, and 34 amino acids (aa), detected by DNA sequence analysis, are expressed early during HCMV infection. In this report, the mRNA start sites for promoters (P1 and P2) were mapped within Towne mtrII by primer extension using RNAs isolated from transformed NIH 3T3 cells. The Towne mtrII promoters exhibited similar activities to the SV40 enhancerless early promoter. Equivalent promoter activities were detected within the mtrII colinear nontransforming region from HCMV strain Tanaka. Two subclones of Towne mtrII (5 440-bp and 3 540-bp), each containing one promoter, were generated utilizing a unique BgII site which also interrupted the 79-aa ORF. In transfection assays, neither the 5 440-bp promoter subclone containing a truncated 79-aa ORF nor the 3 540-bp subclone containing intact 83- and 34-aa ORFs exhibited transforming activity. These data indicated that transformation by HCMV mtrII did not occur by promoter insertion. The identification of these early promoters will allow further studies on the regulation of important HCMV early genes known to be involved in viral/host interactions.

Cell lines containing and expressing the human herpesvirus 6A ts gene are protected from both H- ras and BPV-1 transformation

Human herpesvirus 6A (HHV-6A) strain U1102 was previously shown to contain a 1473u2009bp transformation suppressor gene (ts) (Araujo et al., 1995). Ts inhibited transformation of NIH3T3 cells by H-ras and transcription of the H-ras and human immunodeficiency type 1 (HIV-1) promoters in transient transfection experiments. In the current study, stable NIH3T3 cell lines expressing ts protein were established by transfection with pRc-ts containing the ts gene under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) and a neomycin selectable marker. Selected cell lines contained approximately one to two copies per cell of intact ts sequences, expressed ts protein and grew at approximately the same rate as parental NIH3T3 cells. These cell lines were protected from H-ras transformation while parental and NIH3T3 cells containing the ts gene cloned in the antisense orientation were not. Expression of the chloramphenicol acetyl transferase (CAT) gene under the control of the EJ-H-ras promoter was also suppressed in the ts cell lines but not when the CAT gene was under the control of the murine osteosarcoma virus LTR or human cytomegalovirus immediate early promoter. When NIH3T3 cell lines expressing ts protein were established by infection with the retrovirus, LNCts, the cells expressed ts protein and were protected from H-ras transformation. Furthermore, bovine papillomavirus type 1 (BPV-1) transformation was also suppressed in cells co-transfected with BPV-1 plus ts and in ts expressing cell lines transfected with BPV-1. The BPV-1 p89 and p2443 promoters were down-regulated in 3T3-ts lines. Because the human papillomavirus type 16 (HPV-16) p97 promoter has similarity to the BPV-1 p89 promoter, the ability of ts to suppress p97 was also tested. Like the H-ras and BPV-1 promoters, HPV-16 p97 was down-regulated in 3T3-ts lines. The data indicate the utility of ts against H-ras, BPV-1 and HPV-16 promoters and their respective oncogenes.

Localization and sequence analysis of morphological transforming region III within human cytomegalovirus strain Towne.

A 7.6-kbp BamHI/XbaI EJ subfragment of the Towne XbaI-E fragment of human cytomegalovirus (HCMV) strain Towne has been previously designated as morphological transforming region III (mtrIII) because it induced focal and tumorigenic transformation of rodent fibroblasts. However, in two separate cell systems, mtrIII sequences were not retained because they were not detected in either the focal, tumor or tumor-derived cell lines. In this report, mtrIII was localized to a 2.1-kbp SalI/XbaI DNA fragment. The sequence of the 2,085-bp region was determined and compared to the colinear DNA from HCMV strain AD169. DNA sequence analysis revealed the presence of five open reading frames in Towne mtrIII, two of which are conserved in strain AD169. The localization and sequence analysis of mtrIII will allow further investigation as to the mechanism(s) by which HCMV may play a role in human cancer.