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trans-Activation of viral enhancers including long terminal repeat of the human immunodeficiency virus by the hepatitis B virus X protein
VIROLOGY
169, 479-484 (1989)
trans-Activation of Viral Enhancers Including Long Terminal Repeat of the Human Immunodeficiency Virus by the Hepatitis B Virus X Protein ALEEM SIDDIQUI * I RICHARD GAYNOR,t A . SRINIVASAN,# JOHN MAPOLES,§ AND R . WESLEY FARR *'§ `Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado 80262 ; tDepartment of Hematology-Oncology, UCLA, School of Medicine, Los Angeles, California 90024 ; #CDC, Atlanta, Georgia 30333 ; and §Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262 Received September 14, 1988 ; accepted November 23, 1988 Human hepatitis B virus contains an open reading frame designated X . We have investigated the trans-activating function of the hepatitis B virus X gene in regulating transcriptional control elements . In the HBV genome the major target for X trans-activation is the enhancer element . Further, the X protein stimulates several other viral promoters/ enhancers including the long terminal repeats (LTR) of human retroviruses . One of the viral sequences studied in detail is the human immunodeficiency viral (HIV) LTR which is trans-activated by the X protein . Using mutational analysis of the HIV LTR, we show that the NF-al sequences contained within the U3 region are involved in this stimulatory activity . Nuclear run-on analyses support the notion that X-mediated trans-activation occurs at the level of transcription . (91989 Academic Press, Inc .
promoter/enhancer or a heterologous cassette of promoter/enhancer, synthesis of a 16,000-kDa protein immunoprecipitable with sera from hepatitis B patients has been demonstrated (27) . In this report we describe our studies on the trans-acting function of the X protein, Using two reporter genes, the chloramphenicol acetyltransferase (CAT) and the firefly luciferase (4, 9), we have tested the responses of HBV enhancer and several other viral promoters/enhancers to the transactivating function of X . A stably transformed cell line was established by transfection of HepG2 cells (a human hepatoblastoma cell line that is negative for HBV sequences) (12), with the plasmid vector pNET containing the X ORF (26) . This cell line designated as Get represents a collection of G418-resistent clones that are X ORF-positive clones . Get cells expressed X-related mRNA as shown by Northern blot hybridization (Fig, 3C) and X protein as detected by immunofluorescence in both the cytoplasm and the nucleus (data not presented, Hu Ke-Qin & A . Siddiqui, in preparation) . A transient expression assay system was then used to test the trans-activating function of the X gene product . The transfection efficiencies of HepG2 and the Get cell line were compared with a vector containing the SV40 early promoter without the 72-bp repeats which does not respond to trans-activation by X and were found to be identical (Table 1) . We have previously shown that the core gene of HBV is a weak promoter that requires the assistance of the enhancer element for efficient transcription (21) . In
The hepatitis B virus (HBV) causes chronic hepatitis B and the infection has been linked to primary hepatocellular carcinoma (6, 29) . Although the virus has been difficult to propagate in vitro, a substantial amount of information has accumulated largely from molecular cloning, sequencing, and expression of open reading frames (ORFs) of HBV DNA . These studies have led to the identification of transcriptional control elements that regulate the viral gene expression during infection . One such element is an enhancer, which in the HBV genome is strategically located downstream of S ORF, and upstream of the X and the C ORFs (Fig . 1) (24) . There are four ORFs in the HBV genome (Fig . 1) . Of these, the X ORF is quite intriguing . First, it is located in the region of the genome containing the direct repeats (DR) which are involved in genomic integration (6, 29) . A protein with the expected coding capacity of the ORF, i .e ., 16,000 kDa, has not been identified from infected human liver tissue . However, the immunoprecipitation of an X protein, generated through expression in Escherichia colt or mammalian cells, with sera from hepatitis B carriers or liver cancer patients has been documented (15, 27) . This evidence strongly suggests that the X protein may be expressed at some stage of viral infection . We have previously shown that transcription of X ORF is controlled by an independent promoter sequence (27), which may function in conjunction with the upstream enhancer . Using either the homologous To whom requests for reprints should be addressed .
479
0042-6822/89 $3 .00 Copyright © 1989 by Academ,c Press, Inc . All rights of reproduction in any form reserved .
480
SHORT COMMUNICATIONS
NR HIV LTR
Cep site En SPi TATA TAR
CAT or Ludferase
.0.5e me W ,ea 46
FIG . 1 . Genetic map of HBV DNA representing the four open reading frames (ORF) . S, Surface antigen gene : C, core and a antigen gene : X . X gene ; P, putative polymerase ORF ; P1, preS1 promoter, P2, preS2 promoter : Xp, X promoter: Cp, core promoter . pEnS4CAT or pEnS4luc contains an Hpal (970 nt) to Hpall (1312 nt) fragment placed in that orientation in front of an SV40 early promoter (SV40 Ep) without its 72-bp repeat, followed by either CAT or luciferase gene . Human immunodeficiency viral LTR sequences placed in front of CAT or luciferase gene are represented . Also represented are the various domains and 5' deletion mutants .
studying the effect of the X gene product on promoters, we observed that the core promoter activity was stimulated about 5-fold by X in a transient CAT gene expression assay (Fig . 2A) . The stimulation of this activity is further augmented about 10-fold by the presence of an enhancer sequence . Similarly, the activity of the X promoter in conjunction with the enhancer is augmented about 12-fold in Get cells . Since the X promoter and the enhancer share overlapping functional domains, the autoregulatory effect of the X protein on its promoter could not be addressed . The X protein does not seem to directly stimulate the HBV S gene promoters (data not shown) . Next, we studied the effect of X gene expression on the activity of the enhancer in the context of a heterologous promoter, the SV40 early promoter lacking enhancer (72-bp repeat) . We and others (10, 24) have previously shown, by means of gene transfer experiment, that HBV enhancer exhibits cell-type specificity for human liver cells and that this specificity is due to transacting cellular factors . The stimulation of the HBV enhancer activity by the X protein is increased a maximum of 1 0-fold both in the content of the CAT gene in a time-dependent manner (Fig . 2B) and in the luciferase gene (Table 1) . These results demonstrate that the enhancer activity, which shows preference for the liver
cell environment, is further augmented in the presence of X . To determine whether the X gene-mediated transactivation of the HBV enhancer leads to an increased transcription rate of the reporter gene being assayed, we performed a nuclear run-on analysis (2) of Get and HepG2 cells, transiently transfected with the HBV enhancer luciferase plasmid (pEns4luc) (Fig . 1) . Results shown in Fig . 2C clearly indicate that the effect of the X protein is at the level of transcription . Table 1 shows the trans-activation of various viral enhancers by X using the luciferase gene assay system . The activities of HBV enhancer, SV40 enhancer, RSV LTR, and HIV LTR, in response to X, are increased a maximum of 10- to 50-fold, with the SV40 enhancer eliciting the highest response . Two other human retroviral LTRs were also studied . These include HTLV-I and HTLV-II (32) . Figs . 3A and 3B illustrate that these elements are also stimulated by X in a time-dependent manner . The activities of HTLV1 and HIV LTRs were stimulated about 10-fold, the HTLV11 LTR about 4-to 5-fold . We have investigated in detail the effect of X on HIV LTR sequences by making use of 5' deletion mutants (7) in the present analysis . The HIV-linked CAT expression was assayed both in a stably transformed X-expressing Get cell line (Fig, 3C) and in a cotransfection of HepG2 cells with pAdX (27), an X-expressing vector, and pHIV CAT (Fig . 4A) . These data, including those described in Table 1, clearly show a trans-activation of HIV LTR by the HBV gene product . Multiple cis-acting elements of the HIV LTR have been genetically and functionally defined . These include the TAR region (-17 to +80) required for tat . 111 induction, SP1 bindTABLE 1 LUCIFERASE ACTIVITY BY VIRAL PROMOTERS/ENHANCERS IN I IepG2 AND GET CELLS' Light units x 10'/µg protein b Vectors
Promoters/ enhancers
HepG2
Get
pSV232luc pSV21uc pRSVluc pEnS4luc pHIVLTRIuc
SV40 ED SV40 En RSV LTR HBV En HIV LTR
0 .8 2 .2 3 .4 12 .5 6 .0
66 .4 69 .3 97 .0 59 .7
GET is a permanent cell line (uncloned) derived from transfeotion of HepG2 cells with an expression vector . ' The data presented here are averages of luciferase light units obtained from several transfection experiments . Lip, early promoter, En enhancer .
48 1
SHORT COMMUNICATIONS
A HepG2 Get HepG2 Get HepG2 Get I , I , i ,
C Get
HepG2
B min 10 n 16_111n, 39_ .n tin HepG2 Get HepG2 Get HepG2 Get HepG2 Get I I I I I I I I
Vector
C+En p
c p
M
xp+En
Actin
FIG . 2 . Stimulation of CAT activity directed by HBV promoters/enhancer by X protein . Vectors were transiently expressed in the cell lines as indicated . CAT activity was measured by determining the amount of acetylated derivative of chloramphenicol . All CAT assays were performed using the constant amount of cell proteins . Plasmid pSV232luc (4) was used as an internal control in CAT expression experiments to normalize transfections . (A) Cp + En, core promoter and enhancer sequences (Accl fi 8g/ll, 1070 fi 1990) ; Cp, core promoter (Ssrll-Bglll) . Xp + En, X promoter and enhancer (Accl-NCol, 1070 fi 1375) . (B) Time course of CAT activity by pEnS4CAT (Fig . 1) in HepG2 and Get cells . (C) Nuclear transcription analysis of luciferase gene in HepG2 and Get cells after transfection with pFnS4luc . Actin gene was used as an internal control . The vector is p8R322 DNA .
ing sites (-43 to -83), enhancer region (-70 to -160), and putative negative regulatory region (-167 to -218) (7, 18) . The HIV LTR 5' deletion mutants were each deleted to various extent in the 5' region and linked to the CAT gene . These mutants were transfected into Get and HepG2 cells and assayed for stimulation of the CAT gene expression . The mutants HIV LTR-1 7 or -21 which contain the TAR region did not show any stimulation of CAT activity (data not shown) . Similarly, the LTR-46 and LTR-70 mutants failed to show any activa-
about 10-fold (Figs . 4B and 4C), whereas LTR-218 which includes the putative negative regulatory region was about 3- to 4-fold less efficient (Figs . 4B and 4D) . We further tested two point mutants of the HIV LTR, one is in the enhancer sequence repeat (AENH) and the other in the putative negative regulatory region (ANRE), both linked to the CAT gene (J . Garcia and R . Gaynor, unpublished results) . Results shown in Fig . 4E demonstrate that a mutation in the HIV enhancer sequence repeat abolished the stimulatory effect of the HBV X protein . No effect was seen with the NRE mutant . These data localize the HIV enhancer element to be the target of trans-activation by the HBV X protein . Further demonstration of the trans-activating effect of
tion (Fig . 4B) . These results suggest that neither the promoter sequences (TATA and SP 1) nor the TAR region participate in the response to X . The LTR-1 60, on the other hand, displayed maximal induction by X
A 30 min 60 min HepG2 Get HepG2 Get I 1 1 I
B 9 min HepG2 Get I I
D ?11
HepG2
__411L
Get
1
HepG2 I
C
[email protected] Get I
H .pG2 1
Get 1
1
30min 60 min 90 min HepG2 Get HepG2 Get HepG2 Get I I I I I I
FIG . 3. Induction of CAT activity by human retroviral LTR sequences linked to CAT gene in the presence of X protein (Get) . Time course of CAT expression is shown for (A) HTLVI, (B) HTLVII, and (C) HIV LTRs . (D) Northern blot hybridization of 5 mg of poly(A)* RNA purified from GET cells and probed with X ORF-related riboprobe .
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A
HepG2
HepG2 Get I I
-pAOX
.pAdx
C min
70 ^ min HePG2 Get HepG2 Get
HIV LTR
rR-
46
LTP- 70
LIP -160
LTP-218
LTP-160
E HepG2
Get
HepG2 Get
Ftc . 4 . CAT activity by HIV LTR and its 5' deletion mutants in a time-dependent fashion . (A) Time course of CAT activity . (A) HIV LTR in HepG2 and Get cells and in cotransfection with pAdX (27), an X expression vector. (B) LTR-46, LTR-70, LTR-160, and LTR-218 represent deletion mutants in the 5' of HIV LTR shown in Fig . 1 . Time course of (C) LTR-160 and (D) LTR-218 induction of CAT activity by the X protein of HBV . (E) DENH-CAT and ANRE-CAT activity in HepG2 and Get cells . AENH and ANRE represent point mutants of enhancer sequence repeat and the negative regulatory region of the HIV LTR, respectively (1 . Garcia and R . Gaynor, unpublished results) .
the X protein on HIV LTR involved cotransfection of He La cells with pRSX (X ORF is under the Rous sarcoma viral LTR) and the HIV proviral DNA . This cotransfection resulted in a 5-fold increase in the viral titer, assayed by reverse transcriptase activity (Siddiqui and Srinivasan, unpublished results) . These studies are currently being pursued in other cell lines including cells of lymphoid origin . The studies described here and those reported earlier (28, 31) clearly demonstrate that the X gene product has a regulatory role as a transcriptional activator . In the present analysis the viral enhancer elements seem to be the target sites for trans-activation . First, we noted that the X gene product stimulates homologous enhancer using both CAT and the luciferase reporter genes . In the HBV genome, the trans-activating role of X might be more complex . Its strategic location in the genome suggests a pivotal role in the regulation of HBV
gene expression which may occur both at the transcriptional and/or post-transcriptional levels . However, the evidence shown in the present study using a nuclear run-on method indicates its direct transcriptional activation but this result does not exclude an additional post-transcriptional effect, as is seen with the HIV tat . 111 gene product (3, 5, 18, 22) . The regulatory function of X is reminiscent of several known viral regulatory genes which play important regulatory roles in the viral life cycles (30) . By using the deletion mutants, we have localized the target site for X trans-activation on the HIV LTR . The sequences between -80 and -160, which include the HIV enhancer element, appear to be involved in responding to the X protein . The same region of HIV LTR was recently shown to respond to trans-activation by herpes simplex virus (16) . This region contains the two direct repeats of the sequence GGGACTTTCC (the NF-
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SHORT COMMUNICATIONS
lB sequence motif) (33) . To determine the direct involvement of these sequences, a site-directed mutant in the 3G and the 4G sequences of the NF-/B repeats (SENH) repeat was used which exhibited lack of response to the trans-activation by X (Fig . 4E) . Similarly, the 72-bp repeats of SV40 enhancer element (30) are also the target of X trans-activation, whereas the promoter sequences, including the TATA sequence and the SO 1 sites, both in SV40 and the HIV LTR, do not appear to he affected (Table 1 and Fig . 1) . The stimulatory role of HBV X protein reflected in the transcriptional activation of HIV LTR sequences is an interesting observation because of the striking similarities in the epidemiologic pattern of HIV and HBV infections, the mechanics of transmission (through blood and blood products, sexual contact, etc .), the HIV latency, and the possible involvement of cofactors . A high proportion of AIDS patients show serologic evidence of previous infection with HBV (17, 20, 23) . Given the prolonged latency of HIV infection, and the coexistence of these two viruses in the host, it is tempting to implicate HBV as a potential cofactor in contributing to the development of immunodeficiency syndrome . This effect, as we show here, is perhaps mediated by the H BV X protein whose role would be to induce transcriptional activation of HIV LTR, thereby activating a latent HIV infection leading to the manifestation of AIDS . Several other DNA viruses such as HSV, EBV, and CMV have been implicated as trans-activators of HIV (8, 16, 19) . In addressing the mechanism of trans-activation by X, we have demonstrated its effect on transcription by a nuclear transcription ''run-on'' analysis . The subcellular location of X in Get cells was found to be in both the cytoplasm and the nucleus (Hu Ke-Qin and A . Siddiqui, unpublished results) in contrast to a previous study of expression of X ORF in COS cells in which the X protein was localized in the cytoplasm by immunofluorescence (27) . Also in livers infected with HBV, the X protein has been found in both the nucleus and the cytoplasm (Hu Ke-Qin, personal communication) . In this study we have used human liver cells (HepG2) to investigate the trans-activating function of X . In nonliver cells such as monkey or rat fibroblasts, the function of X was less efficient . This implies that the X protein might require certain hepatic proteins in mediating the trans-aclivaring effect . The data presented here support a model of interaction of viral elements with the X protein directly or in combination with other cellular proteins . In investigating the mechanism of trans-activation, it will be important to demonstrate whether X is a DNA-binding protein . Examination of the amino acid sequence of X ORF
does not reveal an acid-rich block of amino acid, although possible zinc finger regions suggestive of DNAbinding proteins can be envisioned (11) . However, one possible way X could activate is by mediating the binding of other cellular proteins to regulatory sequences such as the Ela protein of adenovirus (1) . The understanding of how HBV and HIV could interact with each other in the host requires the demonstration that both viruses functionally coexist in a cell type to bring about the trans-activation proposed in this report . In this respect, there is ample evidence for the presence of HBV DNA in lymphoid cells derived from peripheral blood mononuclear cells and in T cells from AIDS patients (17, 13) . ACKNOWLEDGMENTS We thank Dr. I . Chen and J . Garcia, both of the UCLA School of Medicine, for the generous gifts of HTLV I, HTLV II LTR CAT, and various HIV LTR CAT constructs, respectively . The technical assistance of Peggy Bishop is acknowledged . This work was supported by grants from ACS (NP-586) and NIH (CA33135, A121182) to A .S . A.S . is a recipient of ACS Faculty Research Award .
REFERENCES 1 . BERK, A., Annu. Rev. Genet. 20, 45-79 (1986) . 2 . CLAYTON, D . F,, HARRELSON, A, t ., and DARNED, 1 . JR ., Mol. Cell. Biel. 5, 2623-2632 (1986) . 3 . CULLEN, B . R ., Ce1146, 973-982 (1986) . 4 . DEWET, 1 . R ., WOOD, K. V ., DELUCA, M ., HELINSKI, D . R ., and SuBRAMANI, S ., Mol. Cell. Riot. 7, 725-757 (1987) . 5 . FAUCr. A . S ., Science 239, 617-622 (1988) . 6 . GANEM, D ., and VARMUS, H ., Annu. Rev. Siochem. 56, 651-693 (1987) . 7. GARCIA, I ., Wu, F ., MiTSUVASU, R., and GAYNOR, R . B ., EMI 6, 3761-3770(1987) . 8. GENDELMAN, H ., PHELPS, W ., FEIGENBAUM, L ., OSTROVE, J ., AAcHi . A ., HOWLEY, P . M . . KHOURY, G ., GINSHERC, H ., and MARTIN, M . M ., Proc . Nat?. Acad. Sci USA 83, 9759-9763 (1986) . 9 . GORMAN . C . M ., MOFFAT, L . F ., and HOWARD, B . H ., Mot . Cell. Biol. 2,1044-1051 (1982) . 10 . JAMEEL, S ., and SIDDIOUI, A., Mol. Cep. 8iot. 6,710-715 (1986). 11 . KLUG, A ., and RHODES, D ., T18S 12,464-469 (1987) . 72 . KN0WELS, B ., HOWE, C . C ., and ADEN, D . P., Science 209, 487499(1980) . 13 . LAURE, F., ZAGURY, D ., SAIMOT, A ., GALLO, R . C ., HAHN, B ., and BRECHOT, C ., Science 229, 561-563 (1985) . 14 . MEYERS, M ., TREPO, L. V ., NATH, N ., and SNrNSKY, l . 1 ., L Virot. 57, 101-1 os (1986) . 75 . MORIARrrY, A ., ALEXANDER, H ., LERNER, R ., and THORNTON, G ., Setence 227, 429-433 (1985). 16 . MOSCA, J . D ., BEDNARIK, D . P ., Rw, N . B . K ., ROSEN, C . A ., SocROSKI, 1 . D ., HASELTINE, N . A ., HAYWARD, G . S ., and PITHA, P . M ., Proc . Nail. Acad. Sot . USA 84 .7408-7413(1987) . 17. NOONAN, C ., YOFFE, B ., MANSELL, P ., and MELNICK,1 ., Proc. Natl. Aced. Sci. USA 83, 5698-5702 (1986) . 18 . OKAMOTO, T ., and WONG-STAAL, F,, Ce1147, 29-35 (1986) . 19 . RANDO, R . F ., PELLETT, P . E ., Luclw, P . A ., BOHAN, C . A ., and SRINIVASAN, A ., Oncogene 1, 13-18 (1967) .
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20 . RAVENHOLT, R . T . Lancetii, 885-886 (1983) . 21 . ROOSSINCK, M ., JAMEEL, S ., LOUKIN, S . H ., and SIDDIaui, A ., Mot Cell. B/0I. 6,1393-1400 (1986) . 22. ROSEN, C . A ., SODROSKI, S . G ., GROH, W . C ., DONJTUN, A . I ., LIPPKE, J ., and HASELTINE, W . A ., Nature (London) 3, 555-559 (1986) . 23. RUSTGI, V., HOOFNAGLE, J ., GERIN, J ., GELMANN, E ., REICHERT, C ., COOPER, J ., and MACHER, A ., Ann . Int. Med. 101, 795-797 (1984) . 24. SHAUL, Y., RUTTER, W . J ., and LAUB, D ., EMBO J. 4, 427-430 (1985) . 25. SIDDIoui, A ., Mot. Cell . Blot 3,143 -146 (1983) . 26. SIDOIOUI, A., JAMEEL, S ., and MAPOLES, J . E ., Proc. Natl. Aced. Sci. USA 83, 566-570 (1986) .
27. SIOOIaui, A ., JAMEEL, S ., and MAPOLES, J . E ., Proc . Nat/. Acad. Sci. USA 84,2513-2517 (1987) . 28. SPANDAU, D ., and LEE, C . H ., J Virol. 62, 427-434 (1988) . 29. TIOLLAIS, P ., POURCEL, C ., and DEJEAN, A ., Nature (London) 317, 489-495(1985) . 30 . ToozE, J . "Molecular Biology of Tumor Virus, Part 2 . DNA Tumor Viruses," 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981 . 31 . Twu, J . S ., and SCHLOEMER, R . H . . J. Vlrol. 61, 3448-3453 (1987) . 32. WEISS, R ., TEICH, N ., VARMUS, H ., and COFFIN, J ., "RNA Tumor Viruses," 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985 . 33. WIRTH, T ., and BALTIMORE, D ., EmboJ.7, 3109-3113 .
169, 479-484 (1989)
trans-Activation of Viral Enhancers Including Long Terminal Repeat of the Human Immunodeficiency Virus by the Hepatitis B Virus X Protein ALEEM SIDDIQUI * I RICHARD GAYNOR,t A . SRINIVASAN,# JOHN MAPOLES,§ AND R . WESLEY FARR *'§ `Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado 80262 ; tDepartment of Hematology-Oncology, UCLA, School of Medicine, Los Angeles, California 90024 ; #CDC, Atlanta, Georgia 30333 ; and §Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262 Received September 14, 1988 ; accepted November 23, 1988 Human hepatitis B virus contains an open reading frame designated X . We have investigated the trans-activating function of the hepatitis B virus X gene in regulating transcriptional control elements . In the HBV genome the major target for X trans-activation is the enhancer element . Further, the X protein stimulates several other viral promoters/ enhancers including the long terminal repeats (LTR) of human retroviruses . One of the viral sequences studied in detail is the human immunodeficiency viral (HIV) LTR which is trans-activated by the X protein . Using mutational analysis of the HIV LTR, we show that the NF-al sequences contained within the U3 region are involved in this stimulatory activity . Nuclear run-on analyses support the notion that X-mediated trans-activation occurs at the level of transcription . (91989 Academic Press, Inc .
promoter/enhancer or a heterologous cassette of promoter/enhancer, synthesis of a 16,000-kDa protein immunoprecipitable with sera from hepatitis B patients has been demonstrated (27) . In this report we describe our studies on the trans-acting function of the X protein, Using two reporter genes, the chloramphenicol acetyltransferase (CAT) and the firefly luciferase (4, 9), we have tested the responses of HBV enhancer and several other viral promoters/enhancers to the transactivating function of X . A stably transformed cell line was established by transfection of HepG2 cells (a human hepatoblastoma cell line that is negative for HBV sequences) (12), with the plasmid vector pNET containing the X ORF (26) . This cell line designated as Get represents a collection of G418-resistent clones that are X ORF-positive clones . Get cells expressed X-related mRNA as shown by Northern blot hybridization (Fig, 3C) and X protein as detected by immunofluorescence in both the cytoplasm and the nucleus (data not presented, Hu Ke-Qin & A . Siddiqui, in preparation) . A transient expression assay system was then used to test the trans-activating function of the X gene product . The transfection efficiencies of HepG2 and the Get cell line were compared with a vector containing the SV40 early promoter without the 72-bp repeats which does not respond to trans-activation by X and were found to be identical (Table 1) . We have previously shown that the core gene of HBV is a weak promoter that requires the assistance of the enhancer element for efficient transcription (21) . In
The hepatitis B virus (HBV) causes chronic hepatitis B and the infection has been linked to primary hepatocellular carcinoma (6, 29) . Although the virus has been difficult to propagate in vitro, a substantial amount of information has accumulated largely from molecular cloning, sequencing, and expression of open reading frames (ORFs) of HBV DNA . These studies have led to the identification of transcriptional control elements that regulate the viral gene expression during infection . One such element is an enhancer, which in the HBV genome is strategically located downstream of S ORF, and upstream of the X and the C ORFs (Fig . 1) (24) . There are four ORFs in the HBV genome (Fig . 1) . Of these, the X ORF is quite intriguing . First, it is located in the region of the genome containing the direct repeats (DR) which are involved in genomic integration (6, 29) . A protein with the expected coding capacity of the ORF, i .e ., 16,000 kDa, has not been identified from infected human liver tissue . However, the immunoprecipitation of an X protein, generated through expression in Escherichia colt or mammalian cells, with sera from hepatitis B carriers or liver cancer patients has been documented (15, 27) . This evidence strongly suggests that the X protein may be expressed at some stage of viral infection . We have previously shown that transcription of X ORF is controlled by an independent promoter sequence (27), which may function in conjunction with the upstream enhancer . Using either the homologous To whom requests for reprints should be addressed .
479
0042-6822/89 $3 .00 Copyright © 1989 by Academ,c Press, Inc . All rights of reproduction in any form reserved .
480
SHORT COMMUNICATIONS
NR HIV LTR
Cep site En SPi TATA TAR
CAT or Ludferase
.0.5e me W ,ea 46
FIG . 1 . Genetic map of HBV DNA representing the four open reading frames (ORF) . S, Surface antigen gene : C, core and a antigen gene : X . X gene ; P, putative polymerase ORF ; P1, preS1 promoter, P2, preS2 promoter : Xp, X promoter: Cp, core promoter . pEnS4CAT or pEnS4luc contains an Hpal (970 nt) to Hpall (1312 nt) fragment placed in that orientation in front of an SV40 early promoter (SV40 Ep) without its 72-bp repeat, followed by either CAT or luciferase gene . Human immunodeficiency viral LTR sequences placed in front of CAT or luciferase gene are represented . Also represented are the various domains and 5' deletion mutants .
studying the effect of the X gene product on promoters, we observed that the core promoter activity was stimulated about 5-fold by X in a transient CAT gene expression assay (Fig . 2A) . The stimulation of this activity is further augmented about 10-fold by the presence of an enhancer sequence . Similarly, the activity of the X promoter in conjunction with the enhancer is augmented about 12-fold in Get cells . Since the X promoter and the enhancer share overlapping functional domains, the autoregulatory effect of the X protein on its promoter could not be addressed . The X protein does not seem to directly stimulate the HBV S gene promoters (data not shown) . Next, we studied the effect of X gene expression on the activity of the enhancer in the context of a heterologous promoter, the SV40 early promoter lacking enhancer (72-bp repeat) . We and others (10, 24) have previously shown, by means of gene transfer experiment, that HBV enhancer exhibits cell-type specificity for human liver cells and that this specificity is due to transacting cellular factors . The stimulation of the HBV enhancer activity by the X protein is increased a maximum of 1 0-fold both in the content of the CAT gene in a time-dependent manner (Fig . 2B) and in the luciferase gene (Table 1) . These results demonstrate that the enhancer activity, which shows preference for the liver
cell environment, is further augmented in the presence of X . To determine whether the X gene-mediated transactivation of the HBV enhancer leads to an increased transcription rate of the reporter gene being assayed, we performed a nuclear run-on analysis (2) of Get and HepG2 cells, transiently transfected with the HBV enhancer luciferase plasmid (pEns4luc) (Fig . 1) . Results shown in Fig . 2C clearly indicate that the effect of the X protein is at the level of transcription . Table 1 shows the trans-activation of various viral enhancers by X using the luciferase gene assay system . The activities of HBV enhancer, SV40 enhancer, RSV LTR, and HIV LTR, in response to X, are increased a maximum of 10- to 50-fold, with the SV40 enhancer eliciting the highest response . Two other human retroviral LTRs were also studied . These include HTLV-I and HTLV-II (32) . Figs . 3A and 3B illustrate that these elements are also stimulated by X in a time-dependent manner . The activities of HTLV1 and HIV LTRs were stimulated about 10-fold, the HTLV11 LTR about 4-to 5-fold . We have investigated in detail the effect of X on HIV LTR sequences by making use of 5' deletion mutants (7) in the present analysis . The HIV-linked CAT expression was assayed both in a stably transformed X-expressing Get cell line (Fig, 3C) and in a cotransfection of HepG2 cells with pAdX (27), an X-expressing vector, and pHIV CAT (Fig . 4A) . These data, including those described in Table 1, clearly show a trans-activation of HIV LTR by the HBV gene product . Multiple cis-acting elements of the HIV LTR have been genetically and functionally defined . These include the TAR region (-17 to +80) required for tat . 111 induction, SP1 bindTABLE 1 LUCIFERASE ACTIVITY BY VIRAL PROMOTERS/ENHANCERS IN I IepG2 AND GET CELLS' Light units x 10'/µg protein b Vectors
Promoters/ enhancers
HepG2
Get
pSV232luc pSV21uc pRSVluc pEnS4luc pHIVLTRIuc
SV40 ED SV40 En RSV LTR HBV En HIV LTR
0 .8 2 .2 3 .4 12 .5 6 .0
66 .4 69 .3 97 .0 59 .7
GET is a permanent cell line (uncloned) derived from transfeotion of HepG2 cells with an expression vector . ' The data presented here are averages of luciferase light units obtained from several transfection experiments . Lip, early promoter, En enhancer .
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C Get
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B min 10 n 16_111n, 39_ .n tin HepG2 Get HepG2 Get HepG2 Get HepG2 Get I I I I I I I I
Vector
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FIG . 2 . Stimulation of CAT activity directed by HBV promoters/enhancer by X protein . Vectors were transiently expressed in the cell lines as indicated . CAT activity was measured by determining the amount of acetylated derivative of chloramphenicol . All CAT assays were performed using the constant amount of cell proteins . Plasmid pSV232luc (4) was used as an internal control in CAT expression experiments to normalize transfections . (A) Cp + En, core promoter and enhancer sequences (Accl fi 8g/ll, 1070 fi 1990) ; Cp, core promoter (Ssrll-Bglll) . Xp + En, X promoter and enhancer (Accl-NCol, 1070 fi 1375) . (B) Time course of CAT activity by pEnS4CAT (Fig . 1) in HepG2 and Get cells . (C) Nuclear transcription analysis of luciferase gene in HepG2 and Get cells after transfection with pFnS4luc . Actin gene was used as an internal control . The vector is p8R322 DNA .
ing sites (-43 to -83), enhancer region (-70 to -160), and putative negative regulatory region (-167 to -218) (7, 18) . The HIV LTR 5' deletion mutants were each deleted to various extent in the 5' region and linked to the CAT gene . These mutants were transfected into Get and HepG2 cells and assayed for stimulation of the CAT gene expression . The mutants HIV LTR-1 7 or -21 which contain the TAR region did not show any stimulation of CAT activity (data not shown) . Similarly, the LTR-46 and LTR-70 mutants failed to show any activa-
about 10-fold (Figs . 4B and 4C), whereas LTR-218 which includes the putative negative regulatory region was about 3- to 4-fold less efficient (Figs . 4B and 4D) . We further tested two point mutants of the HIV LTR, one is in the enhancer sequence repeat (AENH) and the other in the putative negative regulatory region (ANRE), both linked to the CAT gene (J . Garcia and R . Gaynor, unpublished results) . Results shown in Fig . 4E demonstrate that a mutation in the HIV enhancer sequence repeat abolished the stimulatory effect of the HBV X protein . No effect was seen with the NRE mutant . These data localize the HIV enhancer element to be the target of trans-activation by the HBV X protein . Further demonstration of the trans-activating effect of
tion (Fig . 4B) . These results suggest that neither the promoter sequences (TATA and SP 1) nor the TAR region participate in the response to X . The LTR-1 60, on the other hand, displayed maximal induction by X
A 30 min 60 min HepG2 Get HepG2 Get I 1 1 I
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__411L
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[email protected] Get I
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30min 60 min 90 min HepG2 Get HepG2 Get HepG2 Get I I I I I I
FIG . 3. Induction of CAT activity by human retroviral LTR sequences linked to CAT gene in the presence of X protein (Get) . Time course of CAT expression is shown for (A) HTLVI, (B) HTLVII, and (C) HIV LTRs . (D) Northern blot hybridization of 5 mg of poly(A)* RNA purified from GET cells and probed with X ORF-related riboprobe .
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A
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Ftc . 4 . CAT activity by HIV LTR and its 5' deletion mutants in a time-dependent fashion . (A) Time course of CAT activity . (A) HIV LTR in HepG2 and Get cells and in cotransfection with pAdX (27), an X expression vector. (B) LTR-46, LTR-70, LTR-160, and LTR-218 represent deletion mutants in the 5' of HIV LTR shown in Fig . 1 . Time course of (C) LTR-160 and (D) LTR-218 induction of CAT activity by the X protein of HBV . (E) DENH-CAT and ANRE-CAT activity in HepG2 and Get cells . AENH and ANRE represent point mutants of enhancer sequence repeat and the negative regulatory region of the HIV LTR, respectively (1 . Garcia and R . Gaynor, unpublished results) .
the X protein on HIV LTR involved cotransfection of He La cells with pRSX (X ORF is under the Rous sarcoma viral LTR) and the HIV proviral DNA . This cotransfection resulted in a 5-fold increase in the viral titer, assayed by reverse transcriptase activity (Siddiqui and Srinivasan, unpublished results) . These studies are currently being pursued in other cell lines including cells of lymphoid origin . The studies described here and those reported earlier (28, 31) clearly demonstrate that the X gene product has a regulatory role as a transcriptional activator . In the present analysis the viral enhancer elements seem to be the target sites for trans-activation . First, we noted that the X gene product stimulates homologous enhancer using both CAT and the luciferase reporter genes . In the HBV genome, the trans-activating role of X might be more complex . Its strategic location in the genome suggests a pivotal role in the regulation of HBV
gene expression which may occur both at the transcriptional and/or post-transcriptional levels . However, the evidence shown in the present study using a nuclear run-on method indicates its direct transcriptional activation but this result does not exclude an additional post-transcriptional effect, as is seen with the HIV tat . 111 gene product (3, 5, 18, 22) . The regulatory function of X is reminiscent of several known viral regulatory genes which play important regulatory roles in the viral life cycles (30) . By using the deletion mutants, we have localized the target site for X trans-activation on the HIV LTR . The sequences between -80 and -160, which include the HIV enhancer element, appear to be involved in responding to the X protein . The same region of HIV LTR was recently shown to respond to trans-activation by herpes simplex virus (16) . This region contains the two direct repeats of the sequence GGGACTTTCC (the NF-
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lB sequence motif) (33) . To determine the direct involvement of these sequences, a site-directed mutant in the 3G and the 4G sequences of the NF-/B repeats (SENH) repeat was used which exhibited lack of response to the trans-activation by X (Fig . 4E) . Similarly, the 72-bp repeats of SV40 enhancer element (30) are also the target of X trans-activation, whereas the promoter sequences, including the TATA sequence and the SO 1 sites, both in SV40 and the HIV LTR, do not appear to he affected (Table 1 and Fig . 1) . The stimulatory role of HBV X protein reflected in the transcriptional activation of HIV LTR sequences is an interesting observation because of the striking similarities in the epidemiologic pattern of HIV and HBV infections, the mechanics of transmission (through blood and blood products, sexual contact, etc .), the HIV latency, and the possible involvement of cofactors . A high proportion of AIDS patients show serologic evidence of previous infection with HBV (17, 20, 23) . Given the prolonged latency of HIV infection, and the coexistence of these two viruses in the host, it is tempting to implicate HBV as a potential cofactor in contributing to the development of immunodeficiency syndrome . This effect, as we show here, is perhaps mediated by the H BV X protein whose role would be to induce transcriptional activation of HIV LTR, thereby activating a latent HIV infection leading to the manifestation of AIDS . Several other DNA viruses such as HSV, EBV, and CMV have been implicated as trans-activators of HIV (8, 16, 19) . In addressing the mechanism of trans-activation by X, we have demonstrated its effect on transcription by a nuclear transcription ''run-on'' analysis . The subcellular location of X in Get cells was found to be in both the cytoplasm and the nucleus (Hu Ke-Qin and A . Siddiqui, unpublished results) in contrast to a previous study of expression of X ORF in COS cells in which the X protein was localized in the cytoplasm by immunofluorescence (27) . Also in livers infected with HBV, the X protein has been found in both the nucleus and the cytoplasm (Hu Ke-Qin, personal communication) . In this study we have used human liver cells (HepG2) to investigate the trans-activating function of X . In nonliver cells such as monkey or rat fibroblasts, the function of X was less efficient . This implies that the X protein might require certain hepatic proteins in mediating the trans-aclivaring effect . The data presented here support a model of interaction of viral elements with the X protein directly or in combination with other cellular proteins . In investigating the mechanism of trans-activation, it will be important to demonstrate whether X is a DNA-binding protein . Examination of the amino acid sequence of X ORF
does not reveal an acid-rich block of amino acid, although possible zinc finger regions suggestive of DNAbinding proteins can be envisioned (11) . However, one possible way X could activate is by mediating the binding of other cellular proteins to regulatory sequences such as the Ela protein of adenovirus (1) . The understanding of how HBV and HIV could interact with each other in the host requires the demonstration that both viruses functionally coexist in a cell type to bring about the trans-activation proposed in this report . In this respect, there is ample evidence for the presence of HBV DNA in lymphoid cells derived from peripheral blood mononuclear cells and in T cells from AIDS patients (17, 13) . ACKNOWLEDGMENTS We thank Dr. I . Chen and J . Garcia, both of the UCLA School of Medicine, for the generous gifts of HTLV I, HTLV II LTR CAT, and various HIV LTR CAT constructs, respectively . The technical assistance of Peggy Bishop is acknowledged . This work was supported by grants from ACS (NP-586) and NIH (CA33135, A121182) to A .S . A.S . is a recipient of ACS Faculty Research Award .
REFERENCES 1 . BERK, A., Annu. Rev. Genet. 20, 45-79 (1986) . 2 . CLAYTON, D . F,, HARRELSON, A, t ., and DARNED, 1 . JR ., Mol. Cell. Biel. 5, 2623-2632 (1986) . 3 . CULLEN, B . R ., Ce1146, 973-982 (1986) . 4 . DEWET, 1 . R ., WOOD, K. V ., DELUCA, M ., HELINSKI, D . R ., and SuBRAMANI, S ., Mol. Cell. Riot. 7, 725-757 (1987) . 5 . FAUCr. A . S ., Science 239, 617-622 (1988) . 6 . GANEM, D ., and VARMUS, H ., Annu. Rev. Siochem. 56, 651-693 (1987) . 7. GARCIA, I ., Wu, F ., MiTSUVASU, R., and GAYNOR, R . B ., EMI 6, 3761-3770(1987) . 8. GENDELMAN, H ., PHELPS, W ., FEIGENBAUM, L ., OSTROVE, J ., AAcHi . A ., HOWLEY, P . M . . KHOURY, G ., GINSHERC, H ., and MARTIN, M . M ., Proc . Nat?. Acad. Sci USA 83, 9759-9763 (1986) . 9 . GORMAN . C . M ., MOFFAT, L . F ., and HOWARD, B . H ., Mot . Cell. Biol. 2,1044-1051 (1982) . 10 . JAMEEL, S ., and SIDDIOUI, A., Mol. Cep. 8iot. 6,710-715 (1986). 11 . KLUG, A ., and RHODES, D ., T18S 12,464-469 (1987) . 72 . KN0WELS, B ., HOWE, C . C ., and ADEN, D . P., Science 209, 487499(1980) . 13 . LAURE, F., ZAGURY, D ., SAIMOT, A ., GALLO, R . C ., HAHN, B ., and BRECHOT, C ., Science 229, 561-563 (1985) . 14 . MEYERS, M ., TREPO, L. V ., NATH, N ., and SNrNSKY, l . 1 ., L Virot. 57, 101-1 os (1986) . 75 . MORIARrrY, A ., ALEXANDER, H ., LERNER, R ., and THORNTON, G ., Setence 227, 429-433 (1985). 16 . MOSCA, J . D ., BEDNARIK, D . P ., Rw, N . B . K ., ROSEN, C . A ., SocROSKI, 1 . D ., HASELTINE, N . A ., HAYWARD, G . S ., and PITHA, P . M ., Proc . Nail. Acad. Sot . USA 84 .7408-7413(1987) . 17. NOONAN, C ., YOFFE, B ., MANSELL, P ., and MELNICK,1 ., Proc. Natl. Aced. Sci. USA 83, 5698-5702 (1986) . 18 . OKAMOTO, T ., and WONG-STAAL, F,, Ce1147, 29-35 (1986) . 19 . RANDO, R . F ., PELLETT, P . E ., Luclw, P . A ., BOHAN, C . A ., and SRINIVASAN, A ., Oncogene 1, 13-18 (1967) .
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20 . RAVENHOLT, R . T . Lancetii, 885-886 (1983) . 21 . ROOSSINCK, M ., JAMEEL, S ., LOUKIN, S . H ., and SIDDIaui, A ., Mot Cell. B/0I. 6,1393-1400 (1986) . 22. ROSEN, C . A ., SODROSKI, S . G ., GROH, W . C ., DONJTUN, A . I ., LIPPKE, J ., and HASELTINE, W . A ., Nature (London) 3, 555-559 (1986) . 23. RUSTGI, V., HOOFNAGLE, J ., GERIN, J ., GELMANN, E ., REICHERT, C ., COOPER, J ., and MACHER, A ., Ann . Int. Med. 101, 795-797 (1984) . 24. SHAUL, Y., RUTTER, W . J ., and LAUB, D ., EMBO J. 4, 427-430 (1985) . 25. SIDDIoui, A ., Mot. Cell . Blot 3,143 -146 (1983) . 26. SIDOIOUI, A., JAMEEL, S ., and MAPOLES, J . E ., Proc. Natl. Aced. Sci. USA 83, 566-570 (1986) .
27. SIOOIaui, A ., JAMEEL, S ., and MAPOLES, J . E ., Proc . Nat/. Acad. Sci. USA 84,2513-2517 (1987) . 28. SPANDAU, D ., and LEE, C . H ., J Virol. 62, 427-434 (1988) . 29. TIOLLAIS, P ., POURCEL, C ., and DEJEAN, A ., Nature (London) 317, 489-495(1985) . 30 . ToozE, J . "Molecular Biology of Tumor Virus, Part 2 . DNA Tumor Viruses," 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981 . 31 . Twu, J . S ., and SCHLOEMER, R . H . . J. Vlrol. 61, 3448-3453 (1987) . 32. WEISS, R ., TEICH, N ., VARMUS, H ., and COFFIN, J ., "RNA Tumor Viruses," 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985 . 33. WIRTH, T ., and BALTIMORE, D ., EmboJ.7, 3109-3113 .