plasmids, Dr. Ken Wilcox for the IE175-monospecific antiserum, and Dr. Pamela ... Ostrove, J. M., Leonard, J., Weck, K. E. & Gendelman, H. E.. (1987) J. Virol.
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9573-9577, November 1991 Medical Sciences
Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus (HIV-1 provirus/tat deletion/transactivation)
WALDEMAR POPIK* AND PAULA M. *Oncology Center and
PITHA*tf
tDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Communicated by Igor Tamm, August 5, 1991 (received for review April 23, 1991)
The herpes simplex virus type 1 (HSV-1)ABSTRACT mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human a2 interferon (H1uIFN-a2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIVal cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIVal cells, HuIFN-a2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIVai cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIVal cells. No major differences could be detected in the expression of HSV-1 immediate-early (LE) genes IE175 and IEIIO (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIVal cells to account for the inability of HSV-1 to induce HIV-1 in HNHIVai cells. However, major differences were observed in the binding pattern of NF-KcB-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-KB-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIVal cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.
The human immunodeficiency virus type 1 (HIV-1) can persist in infected cells in vitro without significant expression. Mosca et al. (1, 2) and others (3-7) have previously shown that transcriptional activity of the HIV-1 long terminal repeat (LTR) can be substantially enhanced by infection with herpes simplex virus type 1 (HSV-1) and that the products of the HSV-1 immediate-early (IE) viral genes designated IEIIO (ICPO) and IE175 (ICP4) are essential for this transactivation. It has been suggested (8) that the opportunistic infection with HSV-1, which can often be demonstrated in HIV-1-infected patients (9), plays a role in the transition from the chronic to acute type of HIV-1 infection. Replication of HIV-1 depends on the function of two accessory genes, tat and rev, which have a critical role in the viral life cycle (10, 11). Although the exact mechanism of Tat-mediated transactivation of expression of HIV-1 provirus remains controversial, the transactivation response element TAR was identified at the 5' terminus of all HIV-1 mRNA species (12), and both transcriptional and posttranscriptional effects of Tat have been described (13, 14). The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9573
Cells containing the integrated tat-defective HIV-1 provirus provide a useful model system of HIV-1 latency (unpublished data). In these cells, the HIV-1 tat- provirus is not expressed constitutively; however, its expression can be efficiently induced by addition of Tat or phorbol 12-myristate 13-acetate (PMA). In the present study, we examined whether infection with HSV-1 could replace the requirement for a functional tat gene and asked two questions. (i) Can infection with HSV-1 activate the expression of HIV-1 tatprovirus? (it) Can this HSV-1-mediated transactivation be down-modulated by interferon (IFN)? We have shown that HSV-1 activated proviral transcription and both spliced and unspliced HIV-1 transcripts could be detected in infected cells. However, HSV-1 did not activate HIV-1 provirus in IFN-producing cells. In contrast to several published reports (15-17), IFN did not down-regulate the expression of HSV-1 JE10 (ICP0) and IE175 (ICP4) genes and proteins in these cells but altered the binding pattern of NF-KB-specific nuclear proteins to the core enhancer element of the HIV-1 LTR. These results suggest a previously unreported mechanism by which IFN may alter the expression of cellular and viral genes.
MATERIALS AND METHODS Cells, Viruses, and IFN Assay. The HNHIVdt4 cell line was obtained by cotransfection of HeLa cells with HIV-1 tatdefective pHXBc2-D-tat-2 proviral DNA [HIV-1 tat-] (18) and pSV2neo plasmid and by selection ofthe transfected cells by their ability to grow in the presence of G418. The singlecell-derived clone, in which expression of HIV-1 tat- provirus was highly inducible by transfection with the tat geneexpressing plasmid (19), was selected for this study. The HNHIVaj cell line contains, in addition to pHXBc2-D-tat-2 DNA, the pBK89 fusion plasmid in which the human a2 interferon (HuIFN-a2) gene was placed under the control of
the HIV-1 LTR (20). For the induction, 5 x 108 plaqueforming units of the wild-type HSV-1 strain MPcl-20 was used per ml. Antiviral activities in supernatants were determined by a cytopathic assay as described (21). Plasmids and Probes. The HIV-1 tat-deleted pHXBc2-Dtat-2 plasmid (18) has a deletion of 125 nucleotides (53335458) that includes only the initiation codon of the tat gene and does not alter the splice acceptor sites for tat (5324), rev (5501 and 5507), and nef/env (5523) (18). The splice donor site at 5591 is also unchanged [numbering according to Felber et al. (22)]. The HIV-1 tat-expression plasmid pIIIextatIII (19) Abbreviations: IFN, interferon; HIV-1, human immunodeficiency virus type 1; HSV-1, herpes simplex virus type 1; HuIFN-a2, human a2 interferon; IE, immediate early; LTR, long terminal repeat; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase; moi, multiplicity of infection. tTo whom reprint requests should be addressed at: The Johns Hopkins University, Oncology Center, 418 North Bond Street, Baltimore, MD 21231.
9574
Medical Sciences: Popik and Pitha
contains the entire tat gene (Sal I to BamHI fragment of pHXBc2) inserted behind the HIV-1 LTR and does not express Rev protein (ref. 23 and unpublished results). The pBK89 fusion plasmid (20) contains the HuIFN-a2 gene under the control of the HIV-1 LTR inserted into a modified pLJ retroviral vector (24, 25). The HIV-1 riboprobe, pJM105, encodes the antisense sequence of the Xba I-Pst I fragment of the pHXBc2 clone. For the S1 nuclease analysis of the unspliced and spliced HIV-1 RNA, the end-labeled 321-base-pair (bp)-long Nar I-Acc I fragment ofthe pHXBc2 clone was used. The BamHI fragment of pXhol-C plasmid [1.7 kilobase (kb)] and a 2.5-kb BamHI-Sal I fragment of pIGA-15 plasmid (containing intact genes for the IEJ75 and IEJIO genes of HSV-1, respectively) (1, 26) were used to prepare the respective DNA probes by the random priming method. RNA and Protein Analyses. RNA was isolated from HSV1-infected cells and controls by a guanidine thiocyanate method (27), and Northern and S1 nuclease analyses were done as described (28, 29). The in vitro transcription assay was done as described (8, 30). HSV-1-specific proteins were analyzed by Western blot analysis as described by Clouse et al. (31) with serum from a HSV-1-seropositive patient or a monospecific antiserum to IE175 protein. Antigen-antibody complexes were detected with 125I-labeled protein A. The chloramphenicol acetyltransferase (CAT) assay was done as described (1). Electrophoretic Mobility Shift Assay and UV-Crossinking. Nuclear protein extracts were prepared by the procedure of Dignam et al. (32). For the binding reactions, nuclear proteins (4 ,g) were incubated with the [a-32P]dATP-labeled synthetic double-stranded oligonucleotide corresponding to the two NF-KB sites of the HIV-1 LTR as described by Sen and Baltimore (33). DNA-protein UV-crosslinking was done with a photoreactive 5-bromo-2'-deoxyuridine-substituted HIV-1 NF-KB probe (34). Nucleoprotein complexes were separated by the gel shift assay, gels were UV-irradiated in situ for 20 min, and the protein-DNA adducts were eluted and analyzed on SDS/iOo polyacrylamide gels.
RESULTS Induction of HIV-1 tatf Provirus by HSV-1 Infection. The HSV-1-mediated induction of HIV-1 provirus was studied in two permanently transfected cell lines, HNHIVdt4 and HNHIVa1. Both cell lines contained an integrated tatdefective provirus. The HNHIVaj cells also contained an integrated HuIFN-a2 gene under control of the HIV-1 LTR (pBK89). S1 nuclease analysis of HIV-1 transcripts in the nucleus and cytoplasm of HNHIVdt4 and HNHIVaj cells showed the presence of two protected fragments 321 and 106 bp long that corresponded to the unspliced and spliced HIV-1 RNA, respectively, in cells transfected with the Tatexpressing plasmid (pllextatlI) but not in the untransfected controls (Fig. 1A). These results indicate that both the HNHIVdt4 and HNHIVa1 cells contain integrated HIV-1 tat- provirus, expression of which can be activated by Tat supplied in trans. To determine whether infection with HSV-1 also activates the expression of HIV-1 tat- provirus, the cells were infected with HSV-1 [multiplicity of infection (moi) = 2.5], and the presence of HIV-1 transcripts in the cytoplasm and nucleus was analyzed by S1 nuclease hybridization. However, the 321- and 106-bp-long protected fragments representing the unspliced and spliced HIV-1 RNA, respectively, were detected only in infected HNHIVdt4 cells (Fig. 18) and not in HNHIVa1 cells. Using the primer extension assay, we further found that the transcripts detected in HNHIVdt4 cells were correctly initiated (data not shown).
Proc. Natl. Acad Sci. USA 88 (1991) A
N
$
B
-N
N
I [1'
%I
S-l[c N ,(N
P) (-c);
( .11
N N
.1, \.\ I{S
I
I(-
N 1(C 'N
:-I::
11)()-
**w.
-
s
:-'* S
FIG. 1. S1 nuclease analysis of HIV-1 transcripts in HSV-1infected cells. Cytoplasmic (lanes C) and nuclear (lanes N) RNAs (15 ,ug) isolated from HNHIVdt4 and HNHIVaj cells either 24 hr after transfection with pIIlextatIIl (A) or 3 hr after infection with HSV-1 (moi = 2.5) (B) were hybridized with the Nar I-Acc I DNA probe (lane P) spanning the first splice donor of HIV-1, digested with S1 nuclease, and electrophoresed in 6% polyacrylamide gel with 8 M urea as described (29). Unspliced (U) and spliced (S) HIV-1 transcripts are represented by 321- and 106-nucleotide protected bands, respectively. Lane Co represents a control for nonspecific hybridization ofthe Nar I-Acc I probe with tRNA. Lane M (molecular mass markers) contains pBR322 Msp I fragments.
Since the difference in the expression of HIV-1 tatprovirus in HSV-1-infected HNHIVdt4 and HNHIVa1 cells could be a function of time and moi, we measured the relative levels of HIV-1 RNA in cells infected with HSV-1 at different moi 2, 4, and 6 hr after infection. In HNHIVdt4 cells, HIV-1 transcripts could be detected as early as 2 hr after infection (Fig. 2A), reached a maximum 4 hr after infection, and then gradually decreased. In HNHIVa1 cells, low levels of HIV-1 transcripts were detected only in cells infected with high multiplicity. Hence, in contrast to Tat, HSV-1-mediated transactivation of HIV-1 provirus was impaired in HNHIVaj cells. In HNHIVa1 cells, infection with HSV-1 activated low levels of HuIFN-a2 mRNA (data not shown) and IFN in the medium (40 units/ml) (Fig. 2A), whereas neither IFN-a mRNAs nor protein was synthesized in HSV-1-infected HNHIVdt4 cells. These data indicate that HSV-1 induced only the exogenous but not the endogenous IFN-a2 gene. To determine whether the inhibition of HSV-1-mediated transactivation of HIV-1 tat- provirus in HNHIVaj cells occurred at the transcriptional or posttranscriptional levels, we eiamined the transcription of HIV-1 tat- provirus (Fig. 2B) in nuclei isolated from HSV-1-infected and uninfected cells. The in vitro labeled HIV-1 RNA was identified by hybridization to immobilized HIV-1 DNA (30). Fig. 2B shows that, while only very low levels of HIV-1 transcripts could be detected in uninfected HNHIVdt4 cells [which may represent the short HIV-1 transcripts found by others in the absence of tat gene product (35)], infection with HSV-1 resulted in an accumulation of HIV-1 transcripts in HNHIVdt4 but not in HNHIVal cells. In contrast, induction with PMA and cycloheximide activated HIV-1 transcription in both types of cells. These data suggest that, in HNHIVa1 cells, HSV-1 is unable to induce efficiently the transcription of HIV-1 tat- provirus.
Medical Sciences: Ak
Popik and Pitha
IIINHIIVclt4
mo]
I
0.1
21.5 0.1
INH-IVa1
2
4
hr
Proc. Natl. Acad. Sci. USA 88 (1991)
0.5
2.>
1
.1
5.
(.5- 2.5.7 0. 1 ).5
2.5
6
6
4 2.5~(1. 1
nioi
FINHIVial
lIN-lIVdt4 4
A hr 0.1
.2.5
0.1
9575
2
6
6
4
-
so.;> 0.>o.1 c.> n2
(0. 1 0. I -
28S
-
1S
.1 U
0. 1 0.
2I
.i
-
9.2 IE~~~~lO sea
~~~~~~~.O
1,4. IE IEI-5
-'Amd-
B Actin w
IFN
w w
W'_ww
1p
--
at_
b
HNHIVdt4 HNHI Val I-
d
-
FISV-1 IFN-a-
-
+
+
±-
-
+
-
-
--
