Characterization of the human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) oncogene, kaposin (ORF K12).

BACKGROUND: Human herpesvirus 8 (HHV-8) has been implicated in the etiology of Kaposi's sarcoma (KS), a highly angiogenic tumor of complex histology, and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's 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. Kaposi's 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 Kaposi's 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. Kaposi's 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 (Kaposi's 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 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The current study is a further characterization of kaposin protein. OBJECTIVES: Characterization 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. DESIGN: The 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)

Human cytomegalovirus and human herpesvirus 6 genes that transform and transactivate.

This review is an update on the transforming genes of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6). Both viruses have been implicated in the etiology of several human cancers. In particular, HCMV has been associated with cervical carcinoma and adenocarcinomas of the prostate and colon. In vitro transformation studies have established three HCMV morphologic transforming regions (mtr), i.e., mtrI, mtrII, and mtrIII. Of these, only mtrII (UL111A) is retained and expressed in both transformed and tumor-derived cells. The transforming and tumorigenic activities of the mtrII oncogene were localized to an open reading frame (ORF) encoding a 79-amino-acid (aa) protein. Furthermore, mtrII protein bound to the tumor suppressor protein p53 and inhibited its ability to transactivate a p53-responsive promoter. In additional studies, the HCMV immediate-early protein IE86 (IE2; UL122) was found to interact with cell cycle-regulatory proteins such as p53 and Rb. However, IE86 exhibited transforming activity in vitro only in cooperation with adenovirus E1A. HHV-6 is a T-cell-tropic virus associated with AIDS-related and other lymphoid malignancies. In vitro studies identified three transforming fragments, i.e., SalI-L, ZVB70, and ZVH14. Of these, only SalI-L (DR7) was retained in transformed and tumor-derived cells. The transforming and tumorigenic activities of SalI-L have been localized to a 357-aa ORF-1 protein. The ORF-1 protein was expressed in transformed cells and, like HCMV mtrII, bound to p53 and inhibited its ability to transactivate a p53-responsive promoter. HHV-6 has also been proposed to be a cofactor in AIDS because both HHV-6 and human immunodeficiency virus type 1 (HIV-1) have been demonstrated to coinfect human CD4(+) T cells, causing accelerated cytopathic effects. Interestingly, like the transforming proteins of DNA tumor viruses such as simian virus 40 and adenovirus, ORF-1 was also a transactivator and specifically up-regulated the HIV-1 long terminal repeat when cotransfected into CD4(+) T cells. Finally, based on the interactions of HCMV and HHV-6 transforming proteins with tumor suppressor proteins, a scheme is proposed for their role in oncogenesis.

Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene.

The recently identified human herpesvirus 8 (HHV-8, or Kaposi's sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi's sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865-1869, 1994; P. S. Moore et al., J. Virol. 70:549-558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0. 7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641-6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715-719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342-346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.

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 1473 bp 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.

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, 95-106, 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 18 h. 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, Hodgkin's and non-Hodgkin's 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.

Human cytomegalovirus mtrII oncoprotein binds to p53 and down-regulates p53-activated transcription.

The 79-amino-acid (79-aa) open reading frame (UL111a) gene within morphological transforming region II (mtrII) of human cytomegalovirus strain Towne has been shown to transform rodent cells in vitro (J. Thompson, J. Doniger, and L. J. Rosenthal, Arch. Virol. 136:161-172, 1994). Moreover, a translation termination linker (TTL) mutant of mtrII that coded for the first 49 aa of mtrII oncoprotein (designated TTL49) was sufficient for malignant transformation, whereas a TTL mutant that coded for the first 24 aa (designated TTL24) was not. The current study demonstrates the binding of mtrII oncoprotein to the tumor suppressor protein p53 both in vivo using transiently transfected cells and in vitro using labeled proteins. Furthermore, the C-terminally truncated mtrII protein TTL49, but not truncated protein TTL24, bound to p53. The mtrII binding domain mapped to the N-terminal region of p53, residues 1 to 106, with a critical region from aa 27 to 44, whereas the p53 binding domain of mtrII protein was the first 49 aa. Furthermore, mtrII inhibited p53-activated transcription, indicating its ability to alter p53-directed cellular regulatory mechanisms. mtrII oncoprotein was detected both in stably transfected NIH 3T3 cell lines and human cytomegalovirus-infected HEL 299 cells (as early as 12 h after infection) in the perinuclear region and in the nucleus. mtrII-transformed cell lines, at both early and late passage, exhibited high levels of p53 with a 15-fold-extended half-life. However, p53-activated transcription was suppressed in these cells in spite of the increased p53 levels. Finally, the results with wild-type mtrII and its TTL mutants with respect to p53 binding, p53-activated transcription, and transforming ability suggest that the mechanism of mtrII transformation is linked to both p53 binding and disruption of p53 cell regulation.

Human herpesvirus 6A ts suppresses both transformation by H-ras and transcription by the H-ras and human immunodeficiency virus type 1 promoters.

Human herpesvirus 6 strain U1102 (HHV-6A) was shown to contain a 1,473-bp functional transformation suppressor gene (ts). ts exhibited 24% identity and 51% similarity to adeno-associated virus type 2 Rep68/78. Like adeno-associated virus type 2 Rep68/78, HHV-6A ts suppressed H-ras transformation of NIH 3T3 cells. Suppression of H-ras transformation was eliminated by translation termination linker mutation at amino acid 25, 125, or 245. These data indicated the importance of the C-terminal portion of the ts protein. H-ras transformation was suppressed by ts only when H-ras was expressed by its endogenous H-ras promoter and not when it was expressed by the heterologous murine osteosarcoma virus long terminal repeat (LTR). Furthermore, ts suppressed chloramphenicol acetyltransferase (CAT) activity when the CAT gene was expressed from the H-ras promoter but not the murine osteosarcoma virus LTR promoter. Taken together, the data showed that ts suppressed H-ras transformation at the level of the H-ras promoter. To further identify the interaction of ts with transcriptional regulatory elements, the human immunodeficiency virus type 1 (HIV-1) LTR was used. This promoter was selected because it has well-defined transcriptional regulatory elements for both basal and activated transcription, because its activity is inhibited by the Rep68/78 gene, and because both HHV-6 and HIV-1 naturally infect CD4+ T cells in vivo and have been shown to infect the same cell in vitro. ts suppressed expression from both wild-type and upstream mutant HIV-1 LTR-CAT constructs. However, downstream HIV-1 TAR mutations reversed ts suppression, indicating that TAR is one of the critical elements involved. The data presented demonstrated that HHV-6A ts functionally suppressed H-ras transformation and HIV-1 LTR expression and thus that it may be useful in future gene therapy.

Transcriptional activation of minimal HIV-1 promoter by ORF-1 protein expressed from the SalI-L fragment of human herpesvirus 6.

The SalI-L fragment of human herpesvirus 6 (HHV-6) strain U1102 transformed rodent cells and transactivated the HIV-1 LTR 10- to 15-fold in both monkey fibroblasts and human T-lymphocytes. In this report, the SalI-L transactivator of the HIV-1 LTR was localized to ORF-1 which codes for a protein of 357 amino acids. To determine if ORF-1 required functional Sp1 binding sites or the TATA box element of HIV-1 LTR for transactivation, 5'-deletion mutants of the HIV-1 LTR were employed. Plasmids pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1(S), a mammalian expression vector containing ORF-1, all transactivated a deletion mutant of HIV-1 LTR lacking functional Sp1 binding sites (CD-54). These studies demonstrate that transactivation occurred in the absence of Sp1 binding sites and required only a minimal HIV-1 promoter which contains the TATA box element. The specificity of the SalI-L transactivator for HIV-1 LTR was demonstrated by its inability to transactivate the human papillomavirus type 16 or 18 early promoters. The ORF-1 gene was cloned into and expressed from the pET17b bacterial expression vector. Purified ORF-1 protein was obtained by ammonium sulfate precipitation, Mono-S chromatography, and anti-T7. Tag immunoaffinity chromatography. Transactivation of the HIV-1 LTR by ORF-1 protein was demonstrated by electroporation studies in vivo and by transcription studies in vitro. To substantiate the putative biological role of ORF-1, pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1 all reactivated tat-defective HIV-1 provirus from latently infected cells expressing CD4. Thus, the data presented suggest that HHV-6 infection could have a cofactor role in the progression of AIDS.

A transforming fragment within the direct repeat region of human herpesvirus type 6 that transactivates HIV-1.

HHV-6 infection has been associated with several malignancies including non-Hodgkin's lymphoma and Hodgkin's disease by the presence of high antibody titer and/or the presence of HHV-6 DNA. To understand their oncogenic potential, SalI restriction fragments from HHV-6 strain U1102 were transfected into NIH3T3 cells to assess transforming ability. A 3.9-kbp SalI-L DNA fragment spanning the junction of the direct repeat left (DRL) and unique long segment (UL) regions of HHV-6 induced foci of morphologically altered cells. The SalI-L transformed NIH3T3 focal lines induced tumors in nude mice within 2 weeks. The retention of HHV-6 specific DNA observed in SalI-L transformed cells and their tumor-derived lines suggest a possible maintenance function. Since both HHV-6 infection as well as transforming fragments from other DNA viruses have been shown to transactivate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), SalI-L was examined for transactivation activity. SalI-L up-regulated HIV-1 LTR CAT 10-15 fold in both monkey CV-1 and human T Jurkat cells. The further study of the SalI-L transforming fragment exhibiting transactivation of HIV-1 LTR will elucidate whether these two activities are encoded by a single gene and will aid in the understanding of the interaction between HHV-6 and HIV-1 as it relates to progression of AIDS and/or AIDS-related malignancies.

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

Human 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-bp BanII/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.

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.

Differences in retention and expression of transfected human cytomegalovirus Towne XbaI-E transforming fragment in human cervical and NIH 3T3 lines.

Human cervical and NIH 3T3 cells were transfected with the XbaI-E-transforming fragment of human cytomegalovirus strain Towne. Southern blot hybridization showed that 3 of 4 transformed NIH 3T3 cell lines retained only the mtrII subfragment of Towne XbaI-E, but not the mtrIII subfragment. Even though mtrII was retained, no viral transcripts were detected. Analysis of genomic DNAs isolated from three independently derived lines of Towne XbaI-E-transfected human exocervical epithelial cells previously immortalized by human papillomavirus type 16 (CX16-2/Towne-E) revealed the retention of both mtrII and mtrIII subfragments of Towne XbaI-E even after greater than 30 subpassages. Southern blot hybridizations indicated the integration and rearrangement of mtrII as well as mtrIII. Poly (A)+RNA analysis of CX16-2/Towne-E line revealed a 1.9-kb transcript which hybridized to mtr III. In contrast, no viral transcript from the mtrII region was detected in these cells. The pattern of HPV-16 DNA sequences and the profile of RNA transcripts were similar in the parental human exocervical cells (CX16-2) and in the CX16-2/Towne-E cells. Thus far, the CX16-2/Towne-E lines are nontumorigenic in nude mice. This study highlights not only differences in the ability of Towne XbaI-E to transform rodent cells and not human cells but also differences in the retention and expression of mtrII and mtrIII in these cells.

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.

Analysis of peripheral blood lymphocytes of AIDS and high-risk patients for human cytomegalovirus transforming DNA sequences.

Investigation into the presence of human cytomegalovirus (HCMV) transforming mtrII and mtrIII sequences in peripheral blood lymphocyte (PBL) specimens of AIDS and high-risk patients was carried out by nucleic acid hybridization analyses. These probes were selected because they were viral-specific and lacked homology to normal cellular DNAs. In Southern blot hybridizations carried out under stringent conditions, we detected HCMV mtrII sequences associated with the high-molecular-weight DNAs of PBLs in 17 of 37 patients either with AIDS/Kaposi's sarcoma or at high risk for AIDS. In comparison, only 2 of 17 DNA specimens from PBLs of healthy blood donors showed hybridization to mtrII sequences. The inability to detect hybridization to the mtrIII region in most mtrII-positive specimens suggested a specific retention of mtrII sequences. Our study suggests that the retention of mtrII sequences in high molecular weight DNA may constitute a risk factor for the development/progression of AIDS. Alternatively, the retention of mtrII sequences may occur as a result of enhanced HCMV replication in patients with AIDS or at high risk for AIDS.

The human cytomegalovirus mtrII colinear region in strain Tanaka is transformation defective.

The morphological transforming region II (mtrII) of human cytomegalovirus (HCMV) strain Towne has been localized to a 980-base-pair fragment containing three putative open reading frames (ORFs) of 79, 83, and 34 amino acids (aa). In addition, noncoding DNA sequence elements which have the potential to form stem-loop structures were also observed within mtrII. To determine what elements within HCMV Towne mtrII are important in transformation, colinear regions in other HCMV strains (AD169 and Tanaka) were isolated and a comparison of transforming potential was performed. The results indicated that the 2.2-kilobase colinear region in strain AD169 was transforming, whereas the colinear mtrII region in strain Tanaka showed significantly reduced transforming potential. Analysis of the nucleotide sequence data of these colinear regions revealed the presence of the 79-aa ORF in strains Towne and AD169 and its absence in strain Tanaka. In addition, BglII-digested Towne mtrII, which was cleaved within the 79-aa ORF, was shown to display significantly reduced transforming potential. Since the 83- and 34-aa coding sequences were interrupted in both the transforming AD169 colinear region and the nontransforming Tanaka strains, these ORFs were thought not to be important in transformation. Analysis of the stem-loop structures within each of the mtrII colinear regions did not reveal significant changes among the transforming and nontransforming colinear fragments. Thus, the comparative data indicate an important role for the 79-aa ORF in transformation.

Analysis of human KS biopsies and cloned cell lines for cytomegalovirus, HIV-1, and other selected DNA virus sequences.

Investigation into a possible role of several human viruses, including human cytomegalovirus (HCMV), human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), human herpesvirus 6 (HHV6), and Epstein-Barr virus (EBV) in the pathogenesis of Kaposi's sarcoma (KS) has resulted in the lack of an association of these viruses in KS biopsy and cloned KS cell line specimens. Since nearly all patients with KS, including those with HIV-associated KS, have a high incidence of HCMV infection, HCMV has been proposed to be etiologically associated with KS. Moreover, our previous studies showed the retention of HCMV morphological transforming region II (mtrII) in both transformed and tumor-derived cell lines. As a result, we focused on the nucleic acid hybridization analysis of both KS biopsies from AIDS patients as well as cloned KS endothelial cell lines for HCMV mtrII sequences. We also analyzed KS biopsy and KS cloned cell line specimens for HIV-1, HBV, HHV6, and EBV sequences, since these viruses have also been implicated in the etiology of KS. In one set of experiments, Southern blot analysis revealed the presence of HCMV mtrII sequences in two of six KS biopsies; in other experiments, HBV sequences were found in one of seven KS biopsies. No hybridization in any biopsy tissue was detected for HIV-1 DNA sequences. The analysis of six independently derived cloned KS cell lines was next studied. All these lines were negative for hybridization to the HCMV mtrII transforming fragment as well as to subgenomic fragments of HHV6 and EBV.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor progression mediated by two cooperating DNA segments of human cytomegalovirus.

The terminal fragments (EJ and EM) of the XbaI-E transforming segment of human cytomegalovirus can independently induce the tumorigenic conversion of immortalized cells. To study their interaction, Rat-2 cells were transfected singly or with a combination of cloned EJ and EM DNAs. Large transformed foci were induced at a 10-fold higher frequency by EJ plus EM than by either DNA fragment alone. Focus-derived lines transformed by EJ plus EM produced tumors in syngeneic rats at a much faster rate (5 to 7 days) than did cell lines transformed by EJ or EM alone (25 to 35 days). Southern hybridizations showed that EM-homologous DNA was retained, exhibiting a complex pattern of multiple and amplified bands in EJ-plus-EM lines compared to a simple pattern in EM-induced lines. EJ DNA was not detected in the single or double transformants. The levels of p29, a 29-kilodalton transformation-sensitive marker in Rat-2 cells, were decreased 10- to 100-fold in cell lines transformed by EJ or EM fragment alone. Synthesis of p29 was shut off in EJ- plus-EM transformants. These data demonstrate that two unlinked transforming regions of human cytomegalovirus can cooperate to produce an aggressive tumorigenic phenotype.

Localization and DNA sequence analysis of the transforming domain (mtrII) of human cytomegalovirus.

To define the morphological transforming region II (mtrII) of human cytomegalovirus (HCMV), a series of subclones of the Xba I/BamHI fragment EM was constructed in vitro and tested for focus-forming activity and tumorigenicity. A 980-base-pair subclone of fragment EM was identified, and its nucleotide sequence revealed three small open reading frames (ORFs), encoding 79, 83, and 34 amino acid residues. S1 nuclease analysis of HCMV-infected cells identified several distinct early RNA species within mtrII, two of which (P1 and P2) were of particular interest, since the length of the protected DNA fragments would position the 5' end of the RNAs upstream of the open reading frames. In addition, the 980-base-pair transforming sequence revealed DNA elements capable of forming stem-loop structures. Thus the transforming mtrII domain of HCMV strain Towne contains both small open reading frames that are expressed in lytically infected cells and sequences resembling insertion-like structures that may be involved in transformation.

Isolation and partial characterization of nucleocapsid forms from cells infected with human cytomegalovirus strains AD169 and Towne.

We characterized nucleocapsid (NC) forms generated during infection with human cytomegalovirus (HCMV) strains AD169 and Towne. Two morphological forms of NC were isolated by Renografin-76 banding of the nuclear (N) or cytoplasmic (C) fractions of infected cells. SDS-PAGE analysis of the N- and C-NC forms of AD169 and Towne showed the presence of three polypeptides (150, 33, and 30 KD). The C-NC contained five additional polypeptides (260, 112, 110, 68, and 54 KD) with a counterpart in the mature virion. An additional polypeptide of 37 KD (Towne) or 39 KD (AD169) was only present in the B-NC and not in the A-NC, C-NC, or the mature virion. These polypeptides were basic as determined by two-dimensional PAGE analysis. The 37-KD (Towne) and the 39-KD (AD169) polypeptides were phosphorylated. The NC polypeptides were HCMV specific as shown by immunoprecipitation using a pool of HCMV-positive human antisera. The B-NC DNA was shown to have a restriction pattern identical to that of the infectious viral DNA, but no DNA was detected in the A-NC form. Analysis of NC forms by pulse-chase experiments identified a 'pre-NC' peak which may serve as a precursor to the A- and B-NC forms.

Multiple transforming regions of human cytomegalovirus DNA.

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.