Rhoa Effector Activity Modulation of HIV-1 Replication by a Novel

Modulation of HIV-1 Replication by a Novel RhoA Effector Activity Liping Wang, Hangchun Zhang, Patricia A. Solski, Matthew J. Hart, Channing J. Der and Lishan Su This information is current as of September 24, 2021. J Immunol 2000; 164:5369-5374; ; doi: 10.4049/jimmunol.164.10.5369 http://www.jimmunol.org/content/164/10/5369 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Modulation of HIV-1 Replication by a Novel RhoA Effector Activity1

Liping Wang,*‡ Hangchun Zhang,‡§ Patricia A. Solski,†‡ Matthew J. Hart,¶ Channing J. Der,†‡ and Lishan Su2*‡

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or G␣13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity. The Journal of Immunology, 2000, 164: 5369–5374. Downloaded from

he Rho family of small GTPases (RhoA, Rac1, and proteins (12). These include two families of serine/threonine ki- Cdc42) regulates a variety of important cell signaling and nases. The Rho kinase (ROK) and other ROK family kinases (13, T growth control pathways (1–3). In response to extracel- 14) are required for RhoA-mediated cell transformation, SRF ac- lular stimulation, activated Rho GTPases are involved in actin cy- tivation (15), and actin stress ﬁber formation. Protein kinase N and

toskeletal reorganization (4–6), activation of transcription factors its related kinases also interact with GTP-RhoA, but their effector http://www.jimmunol.org/ such as serum response factor (SRF)3 (7) or NF-␬B (8), and cell functions are not clear (16). In addition, several adaptor proteins cycle progression (9, 10). The molecular mechanisms of the pleo- preferentially bind GTP-RhoA. They include rhophilin (17), rho- tropic effects of Rho GTPases are not clear and may reflect the tekin (18), kinectin (19), and citron (20). The specific functions of complex nature of Rho GTPase regulation (3). Like other members the adaptor effectors are not clear. Using RhoA effector domain of the Ras superfamily GTPases, Rho GTPases bind and hydrolyze mutants, it has recently been reported that distinct effectors are GTP, cycling between a biologically active GTP-bound and an involved in RhoA-mediated transformation of NIH3T3 cells, SRF inactive GDP-bound form. GTPase-activating proteins (GAPs) in- activation, and actin stress fiber formation (21, 22). crease the low intrinsic rate of GTP hydrolysis of Rho proteins, In T cells, Rho GTPases have been implicated in T cell devel- by guest on September 24, 2021 thus converting them to the inactive configuration (inhibitors). The opment and T cell activation (23). Rac1 has been implicated in guanine nucleotide dissociation inhibitors bind to Rho proteins and mediating signals from both TCR and costimulatory receptor lock them into their existing nucleotide-bound state, thus acting as CD28 during T cell activation (24). Cdc42 is reported to organize both positive and negative regulators of Rho proteins. A third class actin polarization of T cells toward APC (25), and defects in its of regulatory proteins, guanine nucleotide exchange factors (GEFs; signaling lead to T cell unresponsiveness (26). RhoA has recently also called Dbl family proteins; Refs. 3 and 11), stimulate the been implicated in mediating CD3/CD28 signals to promote IL-2 exchange of GDP for GTP on Rho proteins, thus converting them production (27). Recently, the RhoA GTPase has been shown to into the biologically active forms (activators). promote survival of pro- and early pre-thymocytes and cell cycle In addition, the diverse functions of RhoA are mediated by the progression of late pre-thymocytes (28). association of GTP-bound RhoA with a number of RhoA effector HIV-1 replication is modulated by a number of cellular signaling pathways regulated by both host and viral factors (29). For example, T cell activation is required for efficient HIV-1 replication in resting T cells (30, 31). Activation of transcription factors Departments of *Microbiology and Immunology and †Pharmacology, ‡Lineberger Comprehensive Cancer Center, School of Medicine, and §Department of Epidemiol- such as NF-␬B (32) and NF-ATc (33, 34) leads to enhanced HIV ogy, School of Public Health, University of North Carolina, Chapel Hill, NC 27599; gene expression and replication. Although the Rho GTPases have ¶ and Onyx Pharmaceuticals, Richmond, CA 94806 been implicated in T cell activation (23, 28, 35), little is known Received for publication October 19, 1999. Accepted for publication March 6, 2000. about how Rho GTPases affect HIV-1 replication. The transmem- The costs of publication of this article were defrayed in part by the payment of page brane glycoprotein (TM or gp41) of HIV-1 contains a long cyto- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. plasmic domain (gp41C). Its function is not clear but has been 1 This work was supported in part by a grant from the March of Dimes Basil implicated in regulating HIV-1 replication and cytopathogenicity O’Connor Scholar Award (to L.S.), and by National Institutes of Health Grants (36). We recently demonstrated that gp41C interacted with the AI41356 (to L.S.) and CA63071 (to C.J.D.). C-terminal domain of p115-RhoGEF (37), which is a specific GEF 2 Address correspondence and reprint requests to Dr. Lishan Su, Department of Mi- and activator of the RhoA GTPase (38). We report here that both crobiology and Immunology, 22-048 Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599. E-mail p115-RhoGEF and its specific activator, G␣13, can inhibit HIV-1 address: [email protected] replication. RhoA activation is required for the inhibition of HIV-1 3 Abbreviations used in this paper: SRF, serum response factor; GEF, guanine nu- replication. The RhoA effector activity involved is genetically sep- cleotide exchange factor; RT, reverse transcriptase; ROK, Rho kinase; GPCR, G protein-coupled receptor; GAP, GTPase-activating protein; WT, wild type; CA, con- arable from those involved in transformation of NIH3T3 cells, stitutively active; DH, Dbl homology; PH, pleckstrin homology. activation of SRF, and actin stress fiber formation.

Materials and Methods Reagents, plasmids, and cell lines All p115-RhoGEF derivatives, including p115FL (1-912), p115dN (249- 912), p115dC (249-802), p115dDH (p115 with an internal deletion from 466 to 547, deleting the Dbl homology (DH) domain), p115dPH (1-720, deleting the C terminus and the pleckstrin homology (PH) domain), p115RGS (1-466, deleting both DH and PH domains), were cloned into the pcDNA3 mammalian expression vector as reported (38). The RhoA alleles (wild type (WT), the constitutively active (CA) 63L mutant and its effector domain mutants, and the dominant negative 19N mutant), lacZ, and CA mutant Cdc42 cDNAs were cloned in the pAX142 mammalian expression vector (39). The pNL4-3 plasmid encodes the entire HIV-1 genome DNA in pUC18 (40). The pNL4.Luc.RϪEϪ plasmid was obtained from the Na- tional Institutes of Health (NIH) AIDS Research and Reference Reagent Program (41). Human CD4 cDNA (T4-pMV7; Ref. 42) was also obtained from NIH. The RhoA (63L) effector domain mutants were constructed by site-directed mutagenesis of the RhoA63L gene and all were subcloned in the pcDNA3 vector. 293T and HeLa-MAGI cells (NIH AIDS Research and Reference Re- agent Program; Ref. 43) were maintained in DMEM supplemented with 10% FBS. Jurkat T cells (kindly provided by G. Crabtree, Stanford, CA) were maintained in RPMI 1640 supplemented with 10% FBS. Downloaded from

HIV-1 production and replication in transfected human cells Transient production of HIV-1 was performed by transfecting the HIV-1 provirus NL4-3 (1 ␮g) with pcDNA3 vector (1 ␮g) or p115, G␣13, and RhoA derivatives (1 ␮g) and pAX142-lacZ (0.1 ␮g) in 293T cells in 6-well ␮ plates with LipofectAMINE (or 0.5 g each with Effectene; Qiagen, Santa http://www.jimmunol.org/ Clarita, CA). At 40–50 h after transfection, HIV-1 virions in the culture supernatant were measured by RT or p24 assays (44) and infectious units were determined by titering the supernatant on HeLa-CD4-LTR-lacZ cells (MAGI) as described previously (43). HIV-1 infection assay was performed as follows. T4-pMV7 DNA was transfected into 293T cells with or without p115-RhoGEF (or RhoA). About 30% of transfected cells showed CD4 expression as determined by FACS at 24 h after transfection. NL4-3 viral stocks (50,000 cpm of RT FIGURE 1. Inhibition of HIV-1 replication by p115-RhoGEF. The activity) were used to infect the transfected cells at 24 h after transfection. HIV-1 proviral genome pNL4–3 was cotransfected with pcDNA3 (vector), HIV-1 replication (supernatant RT activity) was measured at 3–4 days

WT (p115FL), or activated the p115-RhoGEF (p115dN) gene into 293T by guest on September 24, 2021 after infection. To analyze HIV-1 gene expression in transfected cells, 1 ␮gof cells. HIV-1 production in the culture medium was measured at 48 h after pNL4.Luc was cotransfected into 293T cells with 1 ␮g of pcDNA3 vector transfection by p24 ELISA (A) or RT assays (B). Infectious units (C) were or 1 ␮g of the p115 (or RhoA) genes. Cell extracts were analyzed at 48 h measured by titration of the culture supernatant on MAGI cells (43). Data after transfection for luciferase activity with a kit (Promega, Madison, WI). shown are representative of at least three independent experiments. In D, The pAX142-lacZ reporter plasmid (39) was included in the transfection HIV-1 infection assay was performed in 293T cells expressing human CD4 mix and ␤-galactosidase activity was measured. The pAX142 promoter has with the p115FL gene or with vector DNA. HIV-1 replication (RT, 103 been reported previously to be unaffected by Dbl family proteins (39, 45). cpm/ml) was measured at 4 days after infection. Data presented represent To analyze HIV-Luc gene expression in human T cells, Jurkat T cells three independent experiments. SDs of duplicate samples are shown as were transfected with the Superfect reagents (Qiagen). The transfection error bars. mix included 0.3 ␮g of pNL4.Luc DNA, 0.3 ␮g pAX142-lacZ, and various amounts (0, 0.3, 0.6, or 0.9 ␮g) of p115FL or CA RhoA DNA. Total DNA was adjusted to 1.5 ␮g with vector DNA in each transfection. Luciferase ␤ and -galactosidase activity was measured at 48 h after transfection. (data not shown). Therefore, p115-RhoGEF inhibited HIV-1 rep- Expression of RhoA (or p115) proteins in transfected cells was confirmed by Western blot assays with RhoA (or p115)-specific Abs (21, 38). lication in both transfection and infection assays. Like all Rho-specific GEFs (or Dbl family proteins; reviewed in Refs. 3 and 11), p115-RhoGEF contains a DH and a PH domain Results and demonstrates transforming activity in NIH3T3 cells. The DH Activation of p115-RhoGEF suppresses HIV-1 replication domain is involved in the nucleotide exchange activity and the PH To test the possibility that p115-RhoGEF is involved in regulating domain determines its membrane association. Both domains are HIV-1 replication, we studied the effect of p115-RhoGEF on required for the transforming activity (11, 46, 47). To define the HIV-1 replication in a transient HIV-1 replication assay. Ectopic function of p115-RhoGEF involved in the inhibition, a number of expression of WT (p115FL) or an active mutant (p115dN; Ref. 38) p115 deletion mutants were tested. The DH domain of p115 is of p115-RhoGEF significantly inhibited HIV-1 replication from a required for the inhibition of HIV-1 replication because deletion of cotransfected HIV-1 genome (Fig. 1, A–C). Both virion-associated the DH domain abolished its ability to inhibit HIV-1 replication p24 Ag (Fig. 1A) and RT activity (Fig. 1B) and infectious unit (Fig. (Fig. 2, A and C). The PH domain was also required since p115- 1C) assays showed a significant reduction (5- to 10-fold) in HIV-1 dPH also lost activity in inhibiting HIV-1 replication. Thus, mem- production. In addition, HIV-1 replication initiated by infection brane association of p115-RhoGEF is required for the activity. The was also inhibited by p115-RhoGEF. When 293T cells expressing RGS domain of p115-RhoGEF, which functions as a GAP to in- CD4 and/or p115-RhoGEF were infected with HIV-1 (NL4-3), hibit its own activator G␣13 (48, 49), was not required for the p115 also showed significant inhibition (ϳ5-fold) of HIV-1 repli- inhibition. To show that the inhibition of HIV-1 replication by cation (Fig. 1D). Similar levels of CD4 expression were detected p115 is not due to its cytotoxicity, a cotransfected pAX142-lacZ in cells cotransfected with T4-pCM7 and vector or p115-RhoGEF reporter gene, which is not affected by Dbl family proteins (39), The Journal of Immunology 5371 Downloaded from http://www.jimmunol.org/

FIGURE 3. Activation of endogenous p115-RhoGEF and RhoA by G␣13 leads to HIV-1 inhibition. A,WTG␣13 or CA G␣13 was cotransfected with pNL4-3 and HIV-1 replication (RT ϫ 103 cpm/ml) was measured. The experiments were performed three times with similar results. Error bars represent SDs. G␣13WT, G␣13CA, and vector indicate samples transfected with WT G␣13, CA G␣13, and pcDNA3 vector, respectively. B, RhoA (19N) counteracted the HIV-1 inhibitory activity of G␣13. pNL4-3 and G␣13CA were cotransfected with the dominant negative

FIGURE 2. The GEF activity of p115-RhoGEF is required for the in- RhoA mutant RhoA (19N). HIV-1 replication (RT ϫ 103 cpm/ml) was by guest on September 24, 2021 hibition. Different mutants of p115-RhoGEF or the FGD1 gene were co- measured. Three independent experiments were performed with similar transfected with the HIV-1 genome (pNL4-3). HIV-1 replication (p24, ng/ results. Error bars indicate SDs. C, Active RhoA proteins inhibit HIV-1 ml) was measured 48 h after transfection (A). At least three independent replication. pNL4-3 was cotransfected with vector, WT RhoA, mutant experiments were performed with similar results. A cotransfected pAX- RhoA (19N), or the CA RhoA (63L) gene and HIV-1 replication was lacZ gene (45) was included as an internal control (B). A number of p115 measured by p24, RT, or infectious units (IU). Relative levels of HIV-1 deletion mutants were tested and their activity in RhoA activation (GEF replication are presented and samples cotransfected with vector DNA were activity and cell transformation; Ref. 38 and unpublished results) and expressed as 100%. Three independent experiments were performed and HIV-1 inhibition are summarized (C). The numbers indicate the amino acid SDs are shown as error bars. positions of the mutants. The N-terminal 250-amino acid residues encode the RGS domain (GAP of G␣13). DH and PH domains are indicated. ϩ, Ͼ75% of WT p115 (FL) activity; Ϫ, Ͻ20% of WT p115 (FL) activity. To further confirm that RhoA signaling is involved in the inhibition, we directly coexpressed different alleles of RhoA with the was not inhibited by p115-RhoGEF (Fig. 2B). This control also HIV-1 genome (pNL4-3) in 293T cells (Fig. 3C). Both the CA showed that transfection efficiency was not affected by p115 and/or active form of RhoA (63L) and the WT RhoA showed significant HIV-1. inhibitory activity (5- to 10-fold) on HIV-1 replication. The neg- To demonstrate that activation of the endogenous p115-Rho- ative mutant of RhoA (19N), however, showed no significant in- GEF can inhibit HIV-1 replication, its specific upstream G protein hibitory activity. activator was expressed in the HIV-1 replication assay. Ectopic expression of both WT and CA G␣13, which specifically activates Both RhoA and p115-RhoGEF inhibit HIV-1 gene expression p115-RhoGEF activity (15), led to inhibition of HIV-1 replication RhoA may affect many steps of HIV-1 replication such as viral (Fig. 3A). Thus, activation of endogenous p115-RhoGEF by its gene expression, virion assembly, and release. To study the mech- upstream G protein also leads to inhibition of HIV-1 replication. anism of HIV-1 inhibition mediated by RhoA, we analyzed reporter gene expression from an HIV-1 genome (pNL4-Luc), which RhoA activity is required for the inhibition of HIV-1 replication lacks a functional env gene and expresses the luciferase gene in the We confirmed that RhoA-dependent signaling was required for nef region of HIV-1 (41). Both p115-RhoGEF and RhoA (63L) inhibition. Thus, the dominant negative mutant of RhoA19N coun- reduced the luciferase gene expression (ϳ5-fold) when coex- teracted the inhibitory activity of G␣13 on HIV-1 replication (Fig. pressed in 293T cells (Fig. 4A). To show that RhoA is specifically 3B). To show the specificity of RhoA19N, a cotransfected control involved, a CA mutant of Cdc42 (a closely related Rho GTPase; pAX142-lacZ reporter gene was not significantly affected by Ref. 39) did not inhibit HIV-1 gene expression (Fig. 4A). Both the RhoA19N (data not shown). RhoA and Cdc42 genes showed significant induction of SRF (Ref. 5372 RhoA-MEDIATED SUPPRESSION OF HIV-1 REPLICATION Downloaded from

FIGURE 4. Both RhoA and p115 inhibit HIV-1 gene expression. A, Inhibition of luciferase gene expression from the pNL4.Luc genome in 293T cells. The pNL4.Luc genome was cotransfected into 293T cells with pcDNA3 vector (vector), p115FL (p115FL), p115dN (p115dN), the CA RhoA (63L), or a CA Cdc42. The level of luciferase expression (relative http://www.jimmunol.org/ light units, RLU) was measured. Mock (pcDNA3 vector only)-transfected cells were used as background. Experiments were performed in duplicate and repeated three times. SDs of duplicate samples are shown as error bars. FIGURE 5. A distinct RhoA effector activity is involved in HIV-1 in- B, Inhibition of HIV-1 gene expression in human T cells. The pNL4.Luc hibition. A, The RhoA effector domain and mutants. Amino acids are des- plasmid was transfected into Jurkat T cells with vector DNA or various ignated with a one-letter code and numbers in the RhoA coding region are amounts of plasmid DNA encoding p115FL or the CA mutant RhoA63L indicated. The mutated amino acids are underlined. RhoA mutants D28N, (RhoA-CA). The experiments were repeated three times with similar V33E, F39L, and D45Q have been reported previously (22). RhoA mutants results. F39L and E40W have also been described previously (21). RhoA mutants D45N and E47M were constructed for this study. B, RhoA protein expression by various RhoA effector mutants. RhoA proteins were detected in by guest on September 24, 2021 transfected 293T cells with a RhoA-specific antiserum. The lower band in 39 and data not shown). Therefore, signaling pathways mediated mock-transfected cells is nonspecific. All RhoA effector domain mutations by RhoA, but not Cdc42, inhibited HIV-1 replication. were in the activated RhoA63L mutant background. The lane marked To demonstrate the inhibitory activity of RhoA and p115 in RhoA indicates samples transfected with the WT RhoA gene. C, Inhibition human T cells, similar transfection experiments were performed in of HIV-1 replication by different RhoA effector mutants. pNL4-3 was co- the Jurkat T cell line with pNL4-Luc. Expression of HIV-encoded transfected with vector or different RhoA mutant derivatives. HIV-1 pro- luciferase was inhibited by coexpression of p115-RhoGEF or the duction was measured by RT or infectious units assays. The relative HIV-1 replication is presented as percentage of vector controls (100%). 63L, CA RhoA (63L) mutant in a dosage-dependent fashion (Fig. 4B). RhoA (63L). Three independent experiments were performed with similar A cotransfected control reporter gene (pAX142-lacZ) was not sig- results. SDs of duplicate samples are shown as error bars. nificantly inhibited, demonstrating the specificity of the inhibition (data not shown). Thus, RhoA activation also inhibited HIV-1 gene expression in human T cells. mation, but still with low or normal activity in SRF activation and A distinct RhoA effector pathway is involved actin stress fiber formation (21, 22), showed significantly reduced inhibition of HIV-1 replication (Fig. 5C). Thus, activation of SRF Effector domain mutants of RhoA have provided useful reagents to (partially) and actin stress fiber formation by RhoA is neither nec- determine whether distinct RhoA-mediated functions are promoted essary nor sufficient to inhibit HIV-1 replication. These results by shared or distinct effector pathways (21, 22). To define the suggest that the HIV-1 inhibitory activity of RhoA is genetically specific RhoA effector activity involved in the inhibition of HIV-1 separable from its activity in transformation of NIH3T3 cells, ac- replication, we constructed a series of effector domain mutants of tivation of SRF, and actin stress fiber formation. A unique RhoA RhoA63L (Fig. 5A). All RhoA mutant genes expressed similar effector pathway, which is defective in the RhoA F39L mutant, is levels of RhoA proteins (Fig. 5B and data not shown). involved in the inhibition. When their activity to inhibit HIV-1 replication was tested, a distinct profile of RhoA mutants was observed (Fig. 5C and Table I). Although it has impaired activity in transformation of NIH3T3 Discussion cells, SRF activation, and actin stress fiber formation (Ref. 21; We report here that HIV-1 replication can be inhibited by p115- Table I and data not shown), the RhoA E40W mutant showed RhoGEF, which is a specific activator of the RhoA GTPase and efficient inhibition of HIV-1 replication (Fig. 5C). Thus, the effec- preferentially expressed in lymphoid tissues (38, 50). Both the up- tor(s) required for these RhoA activities is distinct from the effec- stream activator (G␣13) and downstream effector (RhoA) of p115 tor(s) required to inhibit HIV-1 replication. Furthermore, the RhoA can also down-regulate HIV-1 replication. Furthermore, we F39L mutant, similar to the RhoA E40W mutant in cell transfor- present evidence that a novel RhoA effector activity is involved. The Journal of Immunology 5373

Table I. RhoA functions and effector loop mutants

Actin Stress HIV-1 RhoA Mutanta Fiberb SRFc Transformationd Suppressione

RhoA (63L) ϩϩϩϩ ϩ D28N ϩϩϩϩ ϩ F39L Ϫ(ϩ) ϩϪ Ϫ E40W ϪϪ Ϫ ϩ D45N ϩϩϩϩ ϩ E47M ϩϩϩϩ ϩ

a RhoA(63L) and the mutant derivatives are described in Fig. 6A. b Based on data from two different reports. F39L is partially defective in promoting actin stress ﬁber in one study (21), but is normal in the other (22). c Activation of SRF is measured with a reporter gene under control of the SRF- dependent promoter (Refs. 21 and 22 and data not shown). ϩϩ, 50–100% of wild- type activity; ϩ, 20–50% of wild-type activity; Ϫ, Ͻ20% of wild-type activity. d Transformation of NIH3T3 cells by various RhoA alleles is summarized (Refs. 21 and 22 and data not shown). ϩ, 50–100% of wild-type activity; Ϫ, Ͻ20% of wild-type activity. e Inhibition of HIV-1 replication by RhoA was measured by RT or infectious units assays (Fig. 5C). ϩ, 50–100% of wild-type activity; Ϫ, Ͻ20% of wild-type activity. Downloaded from

These ﬁndings reveal a novel signal transduction pathway that is involved in regulating HIV-1 replication and pathogenesis. FIGURE 6. The RhoA activation pathway and HIV-1 replication. The Inhibition of HIV-1 replication by RhoA may occur at different putative GPCR linking G␣13 and the RhoA-signaling pathway may trigger steps of the HIV-1 gene expression. First, RhoA may coordinate a cascade of events to modulate HIV-1 replication, actin cytoskeletal or-

HIV-1 transcription during infection since RhoA is involved in the ganization (actin fiber), transcription activation (SRF), cell survival, and http://www.jimmunol.org/ regulation of transcription factors such as SRF (7) and NF-␬B (8). growth. Both the F39L and E40W mutants are defective in interacting with This hypothesis agrees with the finding that RhoA inhibited HIV-1 ROK and in promoting transformation of NIH3T3 cells (21). The E40W gene expression (Fig. 4). However, activation of NF-␬B by RhoA mutant is also defective in SRF activation (X). The F39L mutant is only should enhance HIV-1 gene expression (32). Other transcription partially defective in SRF activation (21, 22). The Y effector pathway is factors important for HIV-1 gene expression may be negatively proposed to mediate actin stress fiber formation and is defective in the E40W mutant (21), but functional in the F39L mutant (21, 22). The HIV-1 regulated by RhoA. Alternatively, repression factors may be in- inhibitory activity is defective in the F39L mutant, but functional in the duced by RhoA activation. In addition, posttranscriptional steps E40W mutant. A novel effector (Z) pathway is proposed to mediate the such as RNA stability, splicing, and transport may also be affected HIV-1 inhibitory function. by RhoA. Second, RhoA is involved in regulating cell cycle pro- by guest on September 24, 2021 gression (9, 10, 28). Possible modulation of cell cycle progression by p115-RhoGEF and RhoA may also affect HIV-1 replication in these target cells (51). However, since RhoA appears to demon- inhibitory effect of gp41C on p115-mediated RhoA activation may strate different activities in different cell types, it will be important not play a significant role in HIV-1 production in transfected 293T to study the effects of p115RhoGEF and RhoA in primary T and cells. We are currently investigating the mechanism of the im- macrophage cells. paired replication of the HIV mutants in T cell lines and in the G␣ 13 has recently been identified as an upstream activator of SCID-hu Thy/Liv mouse. p115-RhoGEF (48). Its possible activation by putative G protein- The RhoA activity in inhibiting HIV-1 replication is distinct coupled receptor (GPCRs, including the HIV-1 coreceptors; Ref. from its activities in cell transformation, SRF activation, and actin 52) suggests that extracellular factors (e.g., chemokines and HIV-1 stress fiber formation. As proposed previously (21), the RhoA ef- ␣ env proteins) may modulate the G 13/p115-RhoGEF/RhoA sig- fector ROK is involved in cell transformation and partly in SRF naling cascade to affect HIV-1 replication (Fig. 6). Further eluci- activation and actin stress fiber formation (Fig. 6). Both the F39L ␣ dation of the interaction among the specific GPCR, G 13, and and the E40W mutants have lost their interaction with ROK (21). p115-RhoGEF will provide valuable information about the mech- An additional effector (X, defective in the E40W mutant) is in- anism of possible GPCR-mediated RhoA activation and its effect volved in mediating SRF activation. A distinct effector (Y) is also on HIV-1 replication. proposed to mediate actin stress fiber formation (21, 22). In this The transmembrane glycoprotein (TM or gp41) of HIV-1 con- report, a novel RhoA effector activity (Z) involved in the inhibition tains a long cytoplasmic domain (gp41C). Its function is not clear of HIV-1 replication is proposed. Mutant F39L is defective in the but has been implicated in regulating HIV-1 replication and cyto- putative interaction (or activation) with Z, which will be useful to pathogenicity (36). We recently demonstrated that HIV-1 gp41C define the novel RhoA effector (Z). interacted with the C-terminal domain of p115-RhoGEF and that the interaction led to inhibition of p115 activity. Mutations in gp41C that disrupted the interaction between gp41C and p115 led Acknowledgments to impaired HIV-1 replication in various T cell lines (37). Thus, it We thank Drs. R. Swanstrom, Jenny Ting, Ian Whitehead, and Keith Burr- is possible that one of the functions of HIV-1 gp41C is to coun- idge for discussion and critically reviewing this manuscript. We also thank teract the inhibitory activity of p115/RhoA to enhance viral repli- the NIH AIDS Research and Reference Reagent Program for providing cation. However, the provirus bearing the mutations in gp41C pro- pNL4.Luc.RϪEϪ (N. Landau), MAGI cells (M. Emerman) and T4-pMV7 duced WT levels of HIV virions when transfected in 293T cells (R. Axel), and the University of North Carolina, Capel Hill/Lineberger (37), suggesting that lack of gp41C interaction (and inhibition) Comprehensive Cancer Center nucleic acid and DNA sequencing, flow with p115 had no effect on HIV gene expression. Therefore, the cytometry, and tissue culture core facilities. 5374 RhoA-MEDIATED SUPPRESSION OF HIV-1 REPLICATION

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