Interactions Between Tat and TAR and Human Immunodeficiency Virus Replication Are Facilitated by Human Cyclin T1 but Not Cyclins T2a Or T2b

Virology 255, 182–189 (1999) Article ID viro.1998.9589, available online at http://www.idealibrary.com on

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Provided by Elsevier - Publisher Connector Interactions between Tat and TAR and Human Immunodeficiency Virus Replication Are Facilitated by Human Cyclin T1 but Not Cyclins T2a or T2b

Jo¨rg Wimmer,* Koh Fujinaga,* Ran Taube,* Thomas P. Cujec,* Yuerong Zhu,† Junmin Peng,† David H. Price,† and B. Matija Peterlin*,1

*Howard Hughes Medical Institute, Departments of Medicine, Microbiology, and Immunology, University of California at San Francisco, San Francisco, California 94143-0703; and †Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 Received October 22, 1998; returned to author for revision December 10, 1998; accepted December 24, 1998

The transcriptional transactivator (Tat) from the human immunodeficiency virus (HIV) does not function efficiently in Chinese hamster ovary (CHO) cells. Only somatic cell hybrids between CHO and human cells and CHO cells containing human chromosome 12 (CHO12) support high levels of Tat transactivation. This restriction was mapped to interactions between Tat and TAR. Recently, human cyclin T1 was found to increase the binding of Tat to TAR and levels of Tat transactivation in rodent cells. By combining individually with CDK9, cyclin T1 or related cyclins T2a and T2b form distinct positive transcription elongation factor b (P-TEFb) complexes. In this report, we found that of these three cyclins, only cyclin T1 is encoded on human chromosome 12 and is responsible for its effects in CHO cells. Moreover, only human cyclin T1, not mouse cyclin T1 or human cyclins T2a or T2b, supported interactions between Tat and TAR in vitro. Finally, after introducing appropriate receptors and human cyclin T1 into CHO cells, they became permissive for infection by and replication of HIV. © 1999 Academic Press

INTRODUCTION Tat to TAR and that the central loop in TAR was dispens- able for low levels of transactivation observed in these The transcriptional transactivator (Tat) is essential for cells (Alonso et al., 1994, 1992; Hart et al., 1995; Jones human immunodeficiency virus (HIV) replication. Indeed, and Peterlin, 1994). This finding suggested the existence HIV-1, which lacks the Tat gene, results in little to no of a cellular Tat cofactor encoded on human chromo- expression of viral proteins in cells (Dayton et al., 1986; some 12, which can bind to the central loop in TAR (Hart Fisher et al., 1986; Huet et al., 1989). Tat is unique among et al., 1995). These initial observations were validated transcriptional activators in that it interacts with an RNA structure rather than a DNA sequence. Interactions be- with the identification of human cyclin T1 as a Tat cofac- tween Tat and the 5Ј bulge in the transactivation re- tor (Wei et al., 1998). Cyclin T1 and the cyclin-dependent sponse element (TAR) RNA have been studied exten- kinase 9 (CDK9) form the positive transcription elonga- sively in vitro. However, the central loop in TAR is also tion factor b (P-TEFb), which is required for Tat transac- required for Tat transactivation in vivo. The simplest tivation (Peng et al., 1998). Not only did this human cyclin model that reconciles these findings requires that Tat T1 increase the binding of Tat to TAR in vitro but also its and a cellular protein bind to the 5Ј bulge and central introduction into rodent cells led to higher levels of Tat loop in TAR, respectively. In this scenario, the loop- transactivation (Wei et al., 1998). Although cyclin T1 had binding protein would also be the transcriptional coacti- been mapped to human chromosome 12, these studies vator of Tat. did not exclude the existence of other proteins that might Some of the evidence for this model came from stud- play subservient roles. With the identification of cyclins ies in rodent cells. Tat functions poorly in mouse (NIH- T2a and T2b, which also form different P-TEFb com- 3T3) and hamster (CHO) cells. However, in rodent–hu- plexes (Peng et al., 1998), their contributions to HIV man somatic cell hybrids and in rodent cells, which transcription must be investigated. additionally contain human chromosome 12 (MHC915 Finally, the issue of whether rodent cells can be made and CHO12), levels of Tat transactivation approach those permissive for HIV replication has assumed some impor- observed in human cells (Hart et al., 1989; Newstein et tance. The development of a small animal model of HIV al., 1990). Further dissection of this phenotype revealed replication and possibly AIDS would aid greatly in the that the primary defect in rodent cells was of tethering development of new antiretroviral approaches, including the development of an anti-HIV vaccine. However, many other blocks to HIV replication in rodent cells could exist; 1 To whom reprint requests should be addressed at Mt. Zion, UCSF Gag, Rev, Vif, and Vpr have all been reported to require Box 0703. Fax: (415) 502-1901. E-mail: [email protected]. human coactivators (Braaten et al., 1996; Simon et al.,

1998; Stivahtis et al., 1997; Winslow and Trono, 1993). To address some of these issues, we first determined that cyclins T2a and T2b are not encoded on human chromosome 12 and that they play no role in Tat transactivation. Second, we found that cyclin T1 is the only C-type cyclin that mediates effects of Tat. Third, the rodent cyclin T1 did not support interactions between Tat and TAR. Fi- nally, the expression of human cyclin T1 and viral receptors was sufficient for HIV replication in CHO cells.

RESULTS Cyclin T1, but not cyclins T2a or T2b, are encoded on human chromosome 12 The P-TEFb complex has been implicated strongly in Tat transactivation (Mancebo et al., 1997; Zhu et al., 1997). P-TEFb is composed of CDK9 and one of three cyclins (T1, T2a, or T2b) encoded by two genes (Peng et al., 1998). In HeLa cells, 80% of P-TEFb contains cyclin T1 and 20% contains either cyclin T2a or T2b (Peng et al., 1998). Human chromosome 12 that encodes cyclin T1 has been demonstrated to increase Tat transactivation in rodent cells (Hart et al., 1989). The chromosomal location of the gene encoding cyclins T2a and T2b and the role of these two cyclins in Tat transactivation are not known. To determine whether cyclins T2a and T2b could rescue Tat transactivation in rodent cells, we first examined whether their genes were also located on human chromosome 12. cDNA fragments from cyclins T1, T2a, and FIG. 1. Cyclin T1, but not cyclins T2a and T2b, is encoded on human T2b were used to probe Southern blots of digested chromosome 12. Presented are Southern blots of genomic DNA from genomic DNA from CHO and CHO12 cells (Fig. 1). DNA CHO, CHO12, and 293T cells cut with HindIII and hybridized under from human 293T kidney cells served as the positive stringent conditions with fluorescein-labeled cDNA probes to human control. The positions and lengths of DNA probes are cyclin T1, T2a, and T2b. The regions from cDNAs used as DNA probes presented below the Southern blots, which also reveal are diagrammed below the Southern blots. Note also the identical sequences shared between cyclins T2a and T2b (gray rectangles). the region of identity between cyclins T2a and T2b. Only Cyclins T2a and T2b differ only C-terminal to residues 642. Lanes in cyclin T1 is present on human chromosome 12 (Fig. 1, Southern blots correspond to DNA from CHO, CHO12, and 293T cells. top, lanes 2 and 3). The intensity of the cyclin T1 band in CHO12 cells is about one half that in 293T cells, which is plasmids containing human cyclin T1, Tat, and HIV-1 compatible with the presence of only one human chro- long terminal repeat (LTR) linked to the CAT reporter mosome 12 in CHO12 cells. From these data, we con- gene into CHO and CHO12 cells. If cyclin T1 was clude that only cyclin T1 is encoded on human chromo- solely responsible for human chromosome 12 pheno- some 12 and is present in CHO12 cells. On the other type, then increasing amounts of exogenous human hand, cyclins T2a and T2b hybridize to two fragments of cyclin T1 over that expressed from human chromo- the same molecular mass only in 293T cells (Fig.1, cyclin some 12 should have little to no effect on Tat transac- T2a and T2b panels). Thus cyclins T2a and T2b are tivation. In addition, the expression of only human encoded on a different human chromosome and are not cyclin T1 was expected to achieve levels of transacti- present in CHO12 cells. vation in CHO cells that equal those observed in CHO12 cells. Human cyclin T1 rescues Tat transactivation in CHO Indeed, increasing amounts of human cyclin T1 in- cells creased the activity of Tat only 2-fold in CHO12 cells Although human cyclin T1 can increase Tat transac- (Fig. 2, bottom). In these cells, Tat transactivation rose tivation in rodent cells, it might not be the only cofactor from 45- to 90-fold (Fig. 2, top). In sharp contrast, cyclin of Tat encoded on human chromosome 12; additional T1 increased levels of Tat transactivation up to 8-fold genes could contribute to Tat transactivation in CHO12 over those observed with Tat alone in CHO cells (Fig. cells. To address this issue directly, we cotransfected 2, bottom). In these cells, Tat transactivation increased 184 WIMMER ET AL.

Effects of human cyclin T1 are specific, and human cyclins T2a and T2b do not rescue Tat transactivation in rodent cells It has been reported that these C-type cyclins can increase the expression from other promoters, such as, the cytomegalovirus (CMV) immediate-early promoter, which also directed the synthesis of Tat in our transfected cells (Peng et al., 1998). Although results in CHO and CHO12 cells suggested that effects of cyclin T1 were specific, we wanted to prove formally that increased levels of Tat cannot rescue the rodent defect in CHO cells. To this end, we cotransfected increasing amounts of the Tat effector plasmid with pHIVSCAT into CHO and CHO12 cells. As presented in Fig. 3A, increasing amounts of Tat over 0.1 ␮g had little to no effect on Tat transactivation in CHO and CHO12 cells. Also, under similar conditions, cyclin T1 had a Ͻ2-fold effect on the expression from the CMV promoter that directed the expression of Tat (data not presented). We conclude that data from Fig. 2 reflect effects of cyclin T1 and not nonspecific effects of increased activity of P-TEFb on transcription in general. Having established optimal ratios between cyclin T1 and Tat for Tat transactivation in CHO and CHO12 cells FIG. 2. Human cyclin T1 increases Tat transactivation in CHO cells. and having demonstrated that these effects were spe- Cotransfections of plasmids encoding Tat and HIV-1 LTR linked to the cific, we wanted to determine next whether cyclins T2a CAT reporter gene resulted in 8- and 45-fold transactivation in CHO and and T2b also contributed to the function of Tat in rodent CHO12 cells, respectively. CAT activities obtained with Tat and TatZX, cells. However, as presented in Fig. 3B, only cyclin T1, which contains a missense mutation in Tat (Cujec et al., 1997), were compared (top). On the addition of human cyclin T1, up to 8- and 2-fold but not cyclins T2a or T2b, rescued Tat transactivation in higher levels of Tat transactivation were observed in CHO and CHO12 CHO cells. Neither of these two related cyclins had any cells, respectively (bottom). In the context of increasing amounts of appreciable effect in CHO12 cells (Fig. 3B). Additionally, cyclin T1, plotted are fold-transactivation by Tat (top) and by human coexpression of Tat and both cyclins T2a and T2b in the cyclin T1 and Tat over that observed with Tat alone (bottom). The same cells had no effect (data not presented). Thus not amount of cotransfected Tat plasmid was kept constant at 0.1 ␮g. Data are representative of three transfections performed in triplicate with only is cyclin T1 alone encoded on human chromosome presented SEM values. Calculations of fold-transactivation by cyclin T1 12 but also human cyclin T1 is solely responsible for and Tat over Tat alone were based on mean values obtained from rescuing the rodent defect in Tat transactivation. individual transfections. Only human cyclin T1 supports interactions between Tat and TAR in vitro from 8- to 64-fold (Fig. 2, top). A linear dose response To test the prediction from genetic studies that the curve was observed over a range of concentrations for rodent cyclin T1 should not support interactions between which ratios of cyclin T1 to Tat varied from 1 to 10 (Fig. Tat and TAR, we performed electrophoretic mobility shift 2). At higher concentrations of cyclin T1, a plateau was assays with radiolabeled TAR and relevant proteins ex- reached, and ultimately decreased levels of Tat trans- pressed in Escherichia coli or coupled in vitro transcrip- activation were observed (data not presented). A sim- tion and translation system using the rabbit reticulocyte ilar observation was reported previously for NIH-3T3 lysate. Additionally, we wanted to know whether cyclins cells and probably represents an imbalance of pro- T2a and T2b could interact with Tat and TAR in vitro.As teins that make up P-TEFb (Wei et al., 1998). Impor- presented in Fig. 4, only the addition of human cyclin T1 tantly, the optimal concentration of cyclin T1 was de- to Tat and TAR led to a strong retarded band (lane 2). This termined to be at a 10-fold excess over Tat, where binding required the central loop (Fig. 4, lane 4) and the absolute levels of Tat transactivation were similar be- 5Ј bulge in TAR (data not presented). Importantly, neither tween CHO and CHO12 cells (Figs. 2 and 3B). We rodent cyclin T1 (Fig. 4, lane 6) nor human cyclins T2a conclude that human cyclin T1 rescues Tat transacti- (Fig. 4, lane 10) and T2b (Fig. 4, lane 14) supported vation in CHO cells and is the only relevant cofactor interactions between Tat and TAR in vitro. Under the encoded on human chromosome 12. conditions of our assay, Tat alone did not bind to TAR Tat AND TAR INTERACTIONS AND HIV REPLICATION 185

FIG. 3. Increasing amounts of Tat and the coexpression of cyclins T2a or T2b do not increase Tat transactivation in CHO cells. (A) Increasing amounts of Tat expressed in CHO and CHO12 cells do not increase levels of Tat transactivation. Over a range of concentrations (0.3–1 ␮g) of Tat (also given under the bar graph), Tat transactivation remains constant in CHO (white bars) and CHO12 (black bars) cells. Data are representative of two independent transfections. (B) Only human cyclin T1, but not cyclins T2a or T2b, increase transactivation in CHO cells. At optimal concentrations, which were determined for cyclin T1 (Fig. 2), only cyclin T1 increased Tat transactivation 8-fold in CHO (white bars) and 2-fold in CHO12 (black bars) cells. Cyclins T2a and T2b had no effect. Data are representative of two independent transfections performed in triplicate with presented SEM values.

(Fig. 4, lanes 6, 10, and 14). Because human cyclin T1 cyclin T1 for these studies. The cloning and sequence of contributed equally to Tat transactivation in both mouse this mouse cyclin T1 will be reported elsewhere. We and hamster cells (Wei et al., 1998), we used the mouse conclude that only human cyclin T1 supports interactions between Tat and TAR in vitro and represents the tethering factor for Tat transactivation in vivo.

Cyclin T1 rescues HIV-1 proviral gene expression in CHO cells To determine whether this rescue of Tat transactivation could be extrapolated to increased levels of expression of viral proteins in CHO cells, we cotransfected

plasmids containing the HIV-1 provirus (HIV-1NL4-3) and cyclin T1 into CHO and CHO12 cells. HIV-1NL4-3 represents a T cell tropic virus and infects cells that express CD4 and CXCR4 on the surface (Adachi et al., 1986). These transfections also addressed the question about potential rodent blocks to other viral regulatory, accessory, and structural proteins, that is to Gag, Rev, Vif, and Vpr (Braaten et al., 1996; Simon et al., 1998; Stivahtis et FIG. 4. Human cyclin T1 supports interactions between Tat and TAR in al., 1997; Winslow and Trono, 1993). Supernatents of vitro. Human cyclins T1, T2a, and T2b and the mouse cyclin T1 were transfected cells were harvested 48 h after transfection, incubated with Tat and labeled TAR RNA. Proteins were expressed from E. coli and coupled in vitro transcription and translation systems using the and amounts of p24 Gag and of infectious viral particles rabbit reticulocyte lysate. Cyclin refers to the addition of various cyclins: were assessed by p24 ELISA and the use of Sx22-1 hT1 (human cyclin T1), mT1 (mouse cyclin T1), hT2a (human cyclin T2a), reporter cells, respectively. Sx22-1 cells are derivative of and hT2b (human cyclin T2b). TAR refers to different TAR structures used: HeLa cells, which contain the ␤-galactosidase gene un- ⌬ wt (wild type), loop (TAR deleted in the central loop). The removal of the der the control of the HIV-1 LTR (Fackler et al., 1997). As 5Ј bulge gave results identical to those observed with ⌬loop (data not presented). Tat refers to the presence (ϩ) and absence (Ϫ) of Tat in our presented in Fig. 5A, cyclin T1 rescued HIV-1 gene ex- reactions. TAR ϩ Tat ϩ hT1 indicates the position of the retarded complex pression in CHO cells to levels observed in CHO12 cells in the presence of TAR, Tat, and human cyclin T1. in a dose-dependent manner. Moreover, viral particles 186 WIMMER ET AL.

FIG. 5. CHO cells that express human cyclin T1 support HIV replication and the production of infectious virions from proviral DNA. (A) Levels of p24 increase with the expression of increasing amounts of human cyclin T1 in CHO cells. Plasmids containing the HIV-1NL4-3 provirus and cyclin T1 were cotransfected into CHO (white bars) and CHO12 (black bars) cells. At 2 days later, levels of p24 were measured in the supernatant. Amounts of plasmid DNA are given below the bar graphs in micrograms. Data are representative of three independent experiments performed in triplicate with presented SEM values. (B) Virions produced by CHO and CHO12 cells can infect human indicator Sx22-1 cells. Supernatants from A were used to infect Sx22-1 cells that express CD4 and CXCR4 and contain the HIV-1 LTR linked to the ␤-galactosidase reporter gene. Numbers of blue cells represent foci of infected cells and can be extrapolated to numbers of infectious virions. Data are representative of three independent experiments. produced by CHO12 cells or by CHO cells expressing required due to small numbers of transfected cells bear- human cyclin T1 were fully competent to infect human ing appropriate receptors for HIV. After the infection of Sx22-1 cells (Fig. 5B). We conclude that the lack of CHO or HeLa cells, Jurkat cells and free virus were human cyclin T1 represents the major impediment to the removed by extensive washing, and viral replication was expression of viral genes from the HIV provirus in CHO allowed to proceed for 2 additional days in the adherent cells and that reported blocks to other viral proteins and cells. Amounts of p24 Gag and infectious virus were virion assembly play minor roles. measured as in Fig. 5. No residual virus could be measured in mock-transfected CHO, CHO12, and HeLa cells The addition of human CD4 and chemokine receptors (data not presented). to cyclin T1 renders CHO cells fully competent for In CHO cells expressing human cyclin T1 and CHO12 HIV infection and replication cells, levels of p24 that approximated those found in Having established that proviral replication was re- HeLa cells were observed with HIV-1YU-2 (Fig. 5A). In stored in CHO cells expressing human cyclin T1, we sharp contrast, CHO cells lacking human cyclin T1 did wanted to determine next whether there were additional not replicate HIV-1YU-2 (Fig. 6A). The same phenotype blocks to viral entry and integration in these cells. Thus was observed with the infectivity assay of HIV-1NL4-3 in we investigated whether the lack of human cyclin T1 Sx22-1 cells (Fig. 6B). In this experiment, HeLa cells Ͼ represents the only impediment to the complete replica- produced slightly 2-fold more infectious HIV-1NL4-3 than tive cycle of both T cell and macrophage tropic HIV. To CHO12 cells (Fig. 6B). Again, human cyclin T1 rescued this end, we coexpressed human CD4, CCR5, or CXCR4 viral replication in CHO cells. From these experiments, receptors together with cyclin T1 in CHO and CHO12 we conclude that human cyclin T1 not only rescues Tat cells (Fig. 6). As a control for human cells, we also transactivation but also that the entire intracellular rep- coexpressed these same receptors in HeLa cells. HeLa licative cycle of HIV. When appropriate human receptors cells were chosen because transfection efficiencies of are also provided, in the presence of human cyclin T1, HeLa and CHO cells by electroporation are comparable. HIV can infect and replicate in CHO cells to levels that Additionally, because HeLa cells do not express CD4, are similar to those achieved in human cells. only transfected cells can be infected by HIV. DISCUSSION After transfection, these cells were cocultured with Jurkat cells that were transfected with plasmids that In this study, we demonstrated that only human cyclin contained the complete HIV-1YU-2 or HIV-1NL4-3 provi- T1, but not cyclins T2a and T2b, are encoded on human ruses. HIV-1YU-2 and HIV-1NL4-3 represent macrophage chromosome 12. Only cyclin T1 rescued the restriction of and T cell tropic viruses, respectively (Adachi et al., 1986; Tat transactivation in CHO cells. Furthermore, only hu- Li et al., 1992). They infect cells bearing CD4 and CCR5 man cyclin T1, but not rodent cyclin T1 or human cyclins and CD4 and CXCR4, respectively (Berger, 1997). Cocul- T2a and T2b, supported interactions between Tat and tivation rather than direct infection by viral stocks was TAR in vitro. These interactions depended on the intact Tat AND TAR INTERACTIONS AND HIV REPLICATION 187

FIG. 6. Human CD4, CCR5 or CXCR4, and cyclin T1 permit HIV infection of CHO cells and viral replication to levels that approximate those observed in human cells. (A) Human cyclin T1 is required in addition to human CD4 and CCR5 for productive HIV-1YU-2 infection of CHO cells. CHO, CHO12, and HeLa cells were cotransfected with plasmids containing human CD4, CCR5, and cyclin T1. They were then infected with HIV-1YU-2. Levels of p24 in the supernatant were measured 2 days later. Human cyclin T1 allows for the replication of HIV-1YU-2 in CHO cells that express human CD4 and CCR5 (white bars). HeLa (gray bars) cells allow for slightly higher levels of expression of viral proteins than CHO12 cells (blacks bars). Amounts of plasmids used are given below the bar graphs. Data are representative of two independent experiments. (B) Human CD4, CXCR4, and cyclin T1 are required and sufficient for the full viral replicative cycle of HIV-1NL4-3, resulting in the production of infectious virions in CHO cells. Supernatants from transfected and infected rodent and HeLa cells were transferred to Sx22-1 indicator cells as in Fig. 5B. Infectious virions were observed only in the presence of human cyclin T1 in CHO cells (white bars), and amounts of infectious viruses were 2.5 times lower in CHO12 (black bars) than in HeLa cells (gray bars). central loop in TAR. Finally, CHO cells expressing human similar. Moreover, the introduction of proviruses and hu- cyclin T1 and appropriate viral receptors became per- man cyclin T1 led to the production of high levels of missive for HIV replication. infectious HIV from CHO cells in our transient expression The effects of human cyclin T1 were not on the general assays. Again, human and rodent cells were compara- transcription of cellular or viral genes. A major caveat to ble. We conclude that apart from the lack of appropriate our experiments could have been that increased levels receptors and human cyclin T1, CHO cells are permissive of Tat would by themselves rescue Tat transactivation in for the function of other viral structural, regulatory, and CHO cells. However, although increasing amounts of accessory proteins. Although additional blocks may be human cyclin T1 had a pronounced effect on Tat trans- present in mouse cells, human cyclin T1 will have to be activation, they did not increase levels of Tat in these included in the establishment of any future model of cells. Moreover, increasing the expression of human AIDS in the transgenic mouse. cyclin T1 had little to no effect on Tat transactivation in Our study is compatible with the simple model that CHO12 cells. These data suggest that one copy of hu- one protein serves as both the RNA-tethering and tran- man chromosome 12 provided sufficient levels of human scriptional coactivator of Tat. This protein is human cy- cyclin T1 and that it is the only protein encoded on clin T1. Thus human cyclin T1 must have surfaces that human chromosome 12 that acts as a coactivator of Tat. bind to Tat and TAR. They might or might not be sepa- Because cyclins T2a and T2b alone or in combination rable, and Tat itself provides an additional surface for had no effect on Tat transactivation, they form different interactions with TAR. In this scenario, Tat binds to the 5Ј P-TEFb complexes that are not recruited by Tat to TAR. bulge and human cyclin T1 to the central loop in TAR. These conclusions were strengthened by binding stud- Subsequent to the binding to TAR, CDK9, which is in the ies, in which only human cyclin T1 supported interac- P-TEFb complex with the cyclin T1, phosphorylates the tions between Tat and TAR in vitro. This binding was C-terminal domain of RNA polymerase II. Efficient elon- specific and required an intact central loop and 5Ј bulge gation of viral transcription ensues. In this process, viral in TAR. Thus genetic studies postulating a defect in the specific interactions between Tat and human cyclin T1 tethering of Tat to TAR in rodent cells now found a solid should become targets for possible future therapeutic biochemical foundation. intervention. Furthermore, human cyclin T1 rescued viral replication in CHO cells. Because only few cells were transfected MATERIALS AND METHODS with all plasmids and could be targeted by HIV, relatively Cell culture low levels of viral replication were observed in our infection assays. However, the infectivity of new virions ob- Jurkat, CHO, and CHO12 cells were grown in RPMI tained from human (HeLa) and rodent (CHO) cells was 1640 supplemented with 10% FCS and antibiotics with 188 WIMMER ET AL.

additional 4 mM histidinol (CHO12). HeLa, Sx22-1, and Proviral transfections 293T cells were grown in DMEM with 10% FCS and Cells (107 CHO or CHO12) were cotransfected with 3.5 antibiotics. They were maintained at 37°C and 5% CO . 2 ␮g of pNL4-3 plasmid, which contains the entire

HIV-1NL4-3 provirus, and varying amounts of pcDNA3-T1 Southern blotting by electroporation (250 V, 960 ␮F). The total amount of ␮ Genomic DNA was extracted from CHO, CHO12, and DNA was kept constant at 30 g with the addition of 293T cells using standard procedures. DNA (10 ␮g) was empty pcDNA3 plasmid vector. At 48 h after the trans- digested with HindIII for 12 h at 37°C and subjected to fection, p24 levels were determined by p24 ELISA in the electrophoresis on 0.8% agarose gels. DNA was trans- supernatant (New England Nuclear, Boston, MA). To de- ferred to Hybond Nϩ membranes, cross-linked with UV termine amounts of infectious virions produced by CHO ␮ light, prehybridized for 1 h and hybridized with 10 ng of and CHO12 cells, 500 l of supernatants from these cells fluorescein-labeled DNA probe for 12 h at 65°C following were transferred onto Sx22-1 indicator cells (Fackler et al., 1997), which were seeded at 5 ϫ 104 cells/well in the protocols supplied by the manufacturer (Amersham, 24-well plates the previous day. After another 36 h, these Evanston, IL). Blots were washed twice with 2ϫ SSC, cells were stained for ␤-galactosidase by standard pro- 0.1% Triton X-100, and 0.5ϫ SSC, 0.1% Triton X-100, re- tocols (Fackler et al., 1997). spectively, at 65°C, followed by immunochemical detec- tion and exposure to ECL film (Amersham). DNA probes Viral infection assays were obtained by digesting pcDNA3-T1 with PpuMI and EcoRI and pcDNA3-T2a as well as pcDNA3-T2b with Then, 10 ␮g of pCD4, pcCCR5, pcFusin, and varying PpuMI and ApaI, respectively (Peng et al., 1998). amounts of pcDNA3-T1 were cotransfected into 107 CHO, CHO12, and HeLa cells by electroporation (as above) CAT assays (Deng et al., 1996; Peng et al., 1998). At the same time, 30 ␮g of pYU-2 or pNL4-3 plasmid, which contains the

Cells (CHO and CHO12) were seeded at a density of entire HIV-1YU-2 or HIV-1NL4-3 provirus, were transfected 106 cells/60-mm dish in RPMI and 10% FCS supple- into 107 Jurkat cells by electroporation (180 V, 960 ␮F) mented with antibiotics. After incubation for 48 h, cells (Adachi et al., 1986; Li et al., 1992). After 24 h, transfected were transfected with plasmid DNA using Lipofectamine Jurkat cells were cocultured with target cells and re- (GIBCO BRL, Gaithersburg, MD). The reporter plasmid moved 48 h later by extensive washing. Cells were then pHIVSCAT and the effector plasmid pcDNA3-Tat were grown for additional 48 h, at which time cellular super- described previously (Cujec et al., 1997). All transfections natants were collected and used to determine levels of were balanced for a total 5 ␮g of DNA with the empty p24 or to infect Sx22-1 as described above. When Jurkat pcDNA3 plasmid vector. At 48 h after the transfection, cells were cocultured with untransfected CHO and cells were lysed and CAT activities were measured by a CHO12 cells, no p24 was detected and no infectious liquid scintillation assay as described previously (Cujec virus was recovered. et al., 1997). ACKNOWLEDGMENTS

Electrophoretic mobility shift assays We thank Michael Armanini for his expert secretarial assistance, ␣ 32 Mark Goldsmith and members of his laboratory for advice and exper- The - P-labeled wild-type and mutant TAR RNA spe- tise with viral experiments, Oliver Fackler and Satoshi Kanazawa for cies were prepared by in vitro transcription of linearized technical assistance and comments on the manuscript, and AIDS plasmid templates as described before (Fujinaga et al., Reagents Suppository for reagents. This work was supported by the 1998). Cyclins were synthesized from a coupled in vitro Howard Hughes Medical Institute and National Institutes of Health transcription and translation system using the rabbit re- Grants AI43691 and GM 35500 (D.H.P.). ticulocyte lysate (Promega, WI). Amounts of cyclins were REFERENCES normalized by measuring the incorporation of 35S-methi- onine and cysteine. RNA probes were incubated in bind- Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., ing buffer [30 mM Tris, pH 8.0, 70 mM KCl, 0.1% Nonidet and Martin, M. A. (1986). Production of acquired immunodeficiency ␮ syndrome-associated retrovirus in human and nonhuman cells trans- P-40, 5.5 mM MgCl2, 1 mM DTT, 13% glycerol, 53 g/ml ␮ fected with an infectious molecular clone. J. Virol. 59, 284–291. poly(dI)/poly(dC), 31 g/ml poly(I)/poly(C)] with cyclin Alonso, A., Cujec, T. P., and Peterlin, B. M. (1994). Effects of human proteins (5 ␮l of the IVT product), in the presence or chromosome 12 on interactions between Tat and TAR of human absence of 80 ng of purified Tat protein (Fujinaga et al., immunodeficiency virus type 1. J. Virol. 68, 6505–6513. 1998) for 10 min at 30°C. The reaction mix was subjected Alonso, A., Derse, D., and Peterlin, B. M. (1992). Human chromosome 12 is required for optimal interactions between Tat and TAR of human to nondenaturing polyacrylamide (6%) gel electrophore- immunodeficiency virus type 1 in rodent cells. J. Virol. 66, 4617–4621. sis, and the gel was subsequently dried and analyzed by Berger, E. A. (1997). HIV entry and tropism: The chemokine receptor autoradiography. connection. AIDS 11, Suppl A, S3–S16. Tat AND TAR INTERACTIONS AND HIV REPLICATION 189

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