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. Author manuscript; available in PMC: 2015 May 18.
Published in final edited form as: J Immunol. 2000 May 15;164(10):5369–5374. doi: 10.4049/jimmunol.164.10.5369

Modulation of HIV-1 Replication by a Novel RhoA Effector Activity1

Liping Wang *,‡, Hangchun Zhang ‡,§, Patricia A Solski †,‡, Matthew J Hart , Channing J Der †,‡, Lishan Su *,‡,2
PMCID: PMC4435950  NIHMSID: NIHMS689303  PMID: 10799900

Abstract

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 Gα13, 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 Rho family of small GTPases (RhoA, Rac1, and Cdc42) regulates a variety of important cell signaling and growth control pathways (1–3). In response to extracellular stimulation, activated Rho GTPases are involved in actin cytoskeletal reorganization (4–6), activation of transcription factors such as serum response factor (SRF)3 (7) or NF-κB (8), and cell cycle progression (9, 10). The molecular mechanisms of the pleotropic effects of Rho GTPases are not clear and may reflect the complex nature of Rho GTPase regulation (3). Like other members of the Ras superfamily GTPases, Rho GTPases bind and hydrolyze GTP, cycling between a biologically active GTP-bound and an inactive GDP-bound form. GTPase-activating proteins (GAPs) increase the low intrinsic rate of GTP hydrolysis of Rho proteins, thus converting them to the inactive configuration (inhibitors). The guanine nucleotide dissociation inhibitors bind to Rho proteins and lock them into their existing nucleotide-bound state, thus acting as both positive and negative regulators of Rho proteins. A third class of regulatory proteins, guanine nucleotide exchange factors (GEFs; also called Dbl family proteins; Refs. 3 and 11), stimulate the exchange of GDP for GTP on Rho proteins, thus converting them into the biologically active forms (activators).

In addition, the diverse functions of RhoA are mediated by the association of GTP-bound RhoA with a number of RhoA effector proteins (12). These include two families of serine/threonine kinases. The Rho kinase (ROK) and other ROK family kinases (13, 14) are required for RhoA-mediated cell transformation, SRF activation (15), and actin stress fiber formation. Protein kinase N and its related kinases also interact with GTP-RhoA, but their effector functions are not clear (16). In addition, several adaptor proteins preferentially bind GTP-RhoA. They include rhophilin (17), rho-tekin (18), kinectin (19), and citron (20). The specific functions of the adaptor effectors are not clear. Using RhoA effector domain mutants, it has recently been reported that distinct effectors are involved in RhoA-mediated transformation of NIH3T3 cells, SRF activation, and actin stress fiber formation (21, 22).

In T cells, Rho GTPases have been implicated in T cell development and T cell activation (23). Rac1 has been implicated in mediating signals from both TCR and costimulatory receptor CD28 during T cell activation (24). Cdc42 is reported to organize actin polarization of T cells toward APC (25), and defects in its signaling lead to T cell unresponsiveness (26). RhoA has recently been implicated in mediating CD3/CD28 signals to promote IL-2 production (27). Recently, the RhoA GTPase has been shown to promote survival of pro- and early pre-thymocytes and cell cycle progression of late pre-thymocytes (28).

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 such as NF-κB (32) and NF-ATc (33, 34) leads to enhanced HIV gene expression and replication. Although the Rho GTPases have been implicated in T cell activation (23, 28, 35), little is known about how Rho GTPases affect HIV-1 replication. The transmembrane glycoprotein (TM or gp41) of HIV-1 contains a long cytoplasmic domain (gp41C). Its function is not clear but has been implicated in regulating HIV-1 replication and cytopathogenicity (36). We recently demonstrated that gp41C interacted with the C-terminal domain of p115-RhoGEF (37), which is a specific GEF and activator of the RhoA GTPase (38). We report here that both p115-RhoGEF and its specific activator, Gα13, can inhibit HIV-1 replication. RhoA activation is required for the inhibition of HIV-1 replication. The RhoA effector activity involved is genetically separable from those involved in transformation of NIH3T3 cells, 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.R−E− plasmid was obtained from the National 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 Reagent 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.

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 μg) with pcDNA3 vector (1 μg) or p115, Gα13, and RhoA derivatives (1 μg) and pAX142-lacZ (0.1 μg) in 293T cells in 6-well plates with LipofectAMINE (or 0.5 μg each with Effectene; Qiagen, Santa 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 activity) were used to infect the transfected cells at 24 h after transfection. HIV-1 replication (supernatant RT activity) was measured at 3–4 days after infection.

To analyze HIV-1 gene expression in transfected cells, 1 μg of pNL4.Luc was cotransfected into 293T cells with 1 μg of pcDNA3 vector or 1 μg of the p115 (or RhoA) genes. Cell extracts were analyzed at 48 h after transfection for luciferase activity with a kit (Promega, Madison, WI). The pAX142-lacZ reporter plasmid (39) was included in the transfection mix and β-galactosidase activity was measured. The pAX142 promoter has been reported previously to be unaffected by Dbl family proteins (39, 45).

To analyze HIV-Luc gene expression in human T cells, Jurkat T cells were transfected with the Superfect reagents (Qiagen). The transfection mix included 0.3 μg of pNL4.Luc DNA, 0.3 μg pAX142-lacZ, and various amounts (0, 0.3, 0.6, or 0.9 μg) of p115FL or CA RhoA DNA. Total DNA was adjusted to 1.5 μg with vector DNA in each transfection. Luciferase and β-galactosidase activity was measured at 48 h after transfection.

Expression of RhoA (or p115) proteins in transfected cells was confirmed by Western blot assays with RhoA (or p115)-specific Abs (21, 38).

Results

Activation of p115-RhoGEF suppresses HIV-1 replication

To test the possibility that p115-RhoGEF is involved in regulating HIV-1 replication, we studied the effect of p115-RhoGEF on HIV-1 replication in a transient HIV-1 replication assay. Ectopic expression of WT (p115FL) or an active mutant (p115dN; Ref. 38) of p115-RhoGEF significantly inhibited HIV-1 replication from a cotransfected HIV-1 genome (Fig. 1, A–C). Both virion-associated p24 Ag (Fig. 1A) and RT activity (Fig. 1B) and infectious unit (Fig. 1C) assays showed a significant reduction (5- to 10-fold) in HIV-1 production. In addition, HIV-1 replication initiated by infection was also inhibited by p115-RhoGEF. When 293T cells expressing CD4 and/or p115-RhoGEF were infected with HIV-1 (NL4-3), p115 also showed significant inhibition (~5-fold) of HIV-1 replication (Fig. 1D). Similar levels of CD4 expression were detected in cells cotransfected with T4-pCM7 and vector or p115-RhoGEF (data not shown). Therefore, p115-RhoGEF inhibited HIV-1 replication in both transfection and infection assays.

FIGURE 1.

FIGURE 1

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Inhibition of HIV-1 replication by p115-RhoGEF. The HIV-1 proviral genome pNL4–3 was cotransfected with pcDNA3 (vector), WT (p115FL), or activated the p115-RhoGEF (p115dN) gene into 293T cells. HIV-1 production in the culture medium was measured at 48 h after transfection by p24 ELISA (A) or RT assays (B). Infectious units (C) were measured by titration of the culture supernatant on MAGI cells (43). Data shown are representative of at least three independent experiments. In D, HIV-1 infection assay was performed in 293T cells expressing human CD4 with the p115FL gene or with vector DNA. HIV-1 replication (RT, 103 cpm/ml) was measured at 4 days after infection. Data presented represent three independent experiments. SDs of duplicate samples are shown as error bars.

Like all Rho-specific GEFs (or Dbl family proteins; reviewed in Refs. 3 and 11), p115-RhoGEF contains a DH and a PH domain and demonstrates transforming activity in NIH3T3 cells. The DH domain is involved in the nucleotide exchange activity and the PH domain determines its membrane association. Both domains are required for the transforming activity (11, 46, 47). To define the function of p115-RhoGEF involved in the inhibition, a number of p115 deletion mutants were tested. The DH domain of p115 is required for the inhibition of HIV-1 replication because deletion of the DH domain abolished its ability to inhibit HIV-1 replication (Fig. 2, A and C). The PH domain was also required since p115-dPH also lost activity in inhibiting HIV-1 replication. Thus, membrane association of p115-RhoGEF is required for the activity. The RGS domain of p115-RhoGEF, which functions as a GAP to inhibit its own activator Gα13 (48, 49), was not required for the inhibition. To show that the inhibition of HIV-1 replication by p115 is not due to its cytotoxicity, a cotransfected pAX142-lacZ reporter gene, which is not affected by Dbl family proteins (39), was not inhibited by p115-RhoGEF (Fig. 2B). This control also showed that transfection efficiency was not affected by p115 and/or HIV-1.

FIGURE 2.

FIGURE 2

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The GEF activity of p115-RhoGEF is required for the inhibition. Different mutants of p115-RhoGEF or the FGD1 gene were cotransfected with the HIV-1 genome (pNL4-3). HIV-1 replication (p24, ng/ml) was measured 48 h after transfection (A). At least three independent experiments were performed with similar results. A cotransfected pAX-lacZ gene (45) was included as an internal control (B). A number of p115 deletion mutants were tested and their activity in RhoA activation (GEF activity and cell transformation; Ref. 38 and unpublished results) and HIV-1 inhibition are summarized (C). The numbers indicate the amino acid positions of the mutants. The N-terminal 250-amino acid residues encode the RGS domain (GAP of Gα13). DH and PH domains are indicated. +, >75% of WT p115 (FL) activity; −, <20% of WT p115 (FL) activity.

To demonstrate that activation of the endogenous p115-Rho-GEF can inhibit HIV-1 replication, its specific upstream G protein activator was expressed in the HIV-1 replication assay. Ectopic expression of both WT and CA Gα13, which specifically activates p115-RhoGEF activity (15), led to inhibition of HIV-1 replication (Fig. 3A). Thus, activation of endogenous p115-RhoGEF by its upstream G protein also leads to inhibition of HIV-1 replication.

FIGURE 3.

FIGURE 3

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Activation of endogenous p115-RhoGEF and RhoA by Gα13 leads to HIV-1 inhibition. A, WT Gα13 or CA Gα13 was cotrans-fected with pNL4-3 and HIV-1 replication (RT × 103 cpm/ml) was measured. The experiments were performed three times with similar results. Error bars represent SDs. Gα13WT, Gα13CA, and vector indicate samples transfected with WT Gα13, CA Gα13, and pcDNA3 vector, respectively. B, RhoA (19N) counteracted the HIV-1 inhibitory activity of Gα13. pNL4-3 and Gα13CA were cotransfected with the dominant negative RhoA mutant RhoA (19N). HIV-1 replication (RT × 103 cpm/ml) was measured. Three independent experiments were performed with similar results. Error bars indicate SDs. C, Active RhoA proteins inhibit HIV-1 replication. pNL4-3 was cotransfected with vector, WT RhoA, mutant RhoA (19N), or the CA RhoA (63L) gene and HIV-1 replication was measured by p24, RT, or infectious units (IU). Relative levels of HIV-1 replication are presented and samples cotransfected with vector DNA were expressed as 100%. Three independent experiments were performed and SDs are shown as error bars.

RhoA activity is required for the inhibition of HIV-1 replication

We confirmed that RhoA-dependent signaling was required for inhibition. Thus, the dominant negative mutant of RhoA19N counteracted the inhibitory activity of Gα13 on HIV-1 replication (Fig. 3B). To show the specificity of RhoA19N, a cotransfected control pAX142-lacZ reporter gene was not significantly affected by RhoA19N (data not shown).

To further confirm that RhoA signaling is involved in the inhibition, we directly coexpressed different alleles of RhoA with the HIV-1 genome (pNL4-3) in 293T cells (Fig. 3C). Both the CA active form of RhoA (63L) and the WT RhoA showed significant inhibitory activity (5- to 10-fold) on HIV-1 replication. The negative mutant of RhoA (19N), however, showed no significant inhibitory activity.

Both RhoA and p115-RhoGEF inhibit HIV-1 gene expression

RhoA may affect many steps of HIV-1 replication such as viral gene expression, virion assembly, and release. To study the mechanism of HIV-1 inhibition mediated by RhoA, we analyzed reporter gene expression from an HIV-1 genome (pNL4-Luc), which lacks a functional env gene and expresses the luciferase gene in the nef region of HIV-1 (41). Both p115-RhoGEF and RhoA (63L) reduced the luciferase gene expression (~5-fold) when coexpressed in 293T cells (Fig. 4A). To show that RhoA is specifically involved, a CA mutant of Cdc42 (a closely related Rho GTPase; Ref. 39) did not inhibit HIV-1 gene expression (Fig. 4A). Both the RhoA and Cdc42 genes showed significant induction of SRF (Ref. 39 and data not shown). Therefore, signaling pathways mediated by RhoA, but not Cdc42, inhibited HIV-1 replication.

FIGURE 4.

FIGURE 4

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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 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. B, Inhibition of HIV-1 gene expression in human T cells. The pNL4.Luc plasmid was transfected into Jurkat T cells with vector DNA or various amounts of plasmid DNA encoding p115FL or the CA mutant RhoA63L (RhoA-CA). The experiments were repeated three times with similar results.

To demonstrate the inhibitory activity of RhoA and p115 in human T cells, similar transfection experiments were performed in the Jurkat T cell line with pNL4-Luc. Expression of HIV-encoded luciferase was inhibited by coexpression of p115-RhoGEF or the CA RhoA (63L) mutant in a dosage-dependent fashion (Fig. 4B). A cotransfected control reporter gene (pAX142-lacZ) was not significantly inhibited, demonstrating the specificity of the inhibition (data not shown). Thus, RhoA activation also inhibited HIV-1 gene expression in human T cells.

A distinct RhoA effector pathway is involved

Effector domain mutants of RhoA have provided useful reagents to determine whether distinct RhoA-mediated functions are promoted by shared or distinct effector pathways (21, 22). To define the specific RhoA effector activity involved in the inhibition of HIV-1 replication, we constructed a series of effector domain mutants of RhoA63L (Fig. 5A). All RhoA mutant genes expressed similar levels of RhoA proteins (Fig. 5B and data not shown).

FIGURE 5.

FIGURE 5

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A distinct RhoA effector activity is involved in HIV-1 inhibition. A, The RhoA effector domain and mutants. Amino acids are designated with a one-letter code and numbers in the RhoA coding region are indicated. The mutated amino acids are underlined. RhoA mutants D28N, V33E, F39L, and D45Q have been reported previously (22). RhoA mutants 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 transfected 293T cells with a RhoA-specific antiserum. The lower band in mock-transfected cells is nonspecific. All RhoA effector domain mutations were in the activated RhoA63L mutant background. The lane marked RhoA indicates samples transfected with the WT RhoA gene. C, Inhibition of HIV-1 replication by different RhoA effector mutants. pNL4-3 was cotransfected with vector or different RhoA mutant derivatives. HIV-1 production was measured by RT or infectious units assays. The relative HIV-1 replication is presented as percentage of vector controls (100%). 63L, RhoA (63L). Three independent experiments were performed with similar results. SDs of duplicate samples are shown as error bars.

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 cells, SRF activation, and actin stress fiber formation (Ref. 21; Table I and data not shown), the RhoA E40W mutant showed efficient inhibition of HIV-1 replication (Fig. 5C). Thus, the effector(s) required for these RhoA activities is distinct from the effector(s) required to inhibit HIV-1 replication. Furthermore, the RhoA F39L mutant, similar to the RhoA E40W mutant in cell transformation, but still with low or normal activity in SRF activation and actin stress fiber formation (21, 22), showed significantly reduced inhibition of HIV-1 replication (Fig. 5C). Thus, activation of SRF (partially) and actin stress fiber formation by RhoA is neither necessary nor sufficient to inhibit HIV-1 replication. These results suggest that the HIV-1 inhibitory activity of RhoA is genetically separable from its activity in transformation of NIH3T3 cells, activation of SRF, and actin stress fiber formation. A unique RhoA effector pathway, which is defective in the RhoA F39L mutant, is involved in the inhibition.

Table I.

RhoA functions and effector loop mutants

RhoA Mutanta Actin
Stress Fiberb 		SRFc Transformationd HIV-1
Suppressione
RhoA (63L) + ++ + +
D28N + ++ + +
F39L −(+) + − −
E40W − − − +
D45N + ++ + +
E47M + ++ + +
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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). ++, 50-100% of wild-type activity; +, 20-50% of wild-type activity; −, <20% of wild-type activity.

d

Transformation of NIH3T3 cells by various RhoA alleles is summarized (Refs. 21 and 22 and data not shown). +, 50-100%) of wild-type activity; −, <20%) of wild-type activity.

e

Inhibition of HIV-1 replication by RhoA was measured by RT or infectious units assays (Fig. 5C). +, 50-100%) of wild-type activity; −, <20%) of wild-type activity.

Discussion

We report here that HIV-1 replication can be inhibited by p115-RhoGEF, which is a specific activator of the RhoA GTPase and preferentially expressed in lymphoid tissues (38, 50). Both the upstream activator (Gα13) and downstream effector (RhoA) of p115 can also down-regulate HIV-1 replication. Furthermore, we present evidence that a novel RhoA effector activity is involved. These findings reveal a novel signal transduction pathway that is involved in regulating HIV-1 replication and pathogenesis.

Inhibition of HIV-1 replication by RhoA may occur at different steps of the HIV-1 gene expression. First, RhoA may coordinate HIV-1 transcription during infection since RhoA is involved in the regulation of transcription factors such as SRF (7) and NF-κB (8). This hypothesis agrees with the finding that RhoA inhibited HIV-1 gene expression (Fig. 4). However, activation of NF-κB by RhoA should enhance HIV-1 gene expression (32). Other transcription factors important for HIV-1 gene expression may be negatively regulated by RhoA. Alternatively, repression factors may be induced by RhoA activation. In addition, posttranscriptional steps such as RNA stability, splicing, and transport may also be affected by RhoA. Second, RhoA is involved in regulating cell cycle progression (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 demonstrate different activities in different cell types, it will be important to study the effects of p115RhoGEF and RhoA in primary T and macrophage cells.

Gα13 has recently been identified as an upstream activator of p115-RhoGEF (48). Its possible activation by putative G proteincoupled receptor (GPCRs, including the HIV-1 coreceptors; Ref. 52) suggests that extracellular factors (e.g., chemokines and HIV-1 env proteins) may modulate the Gα13/p115-RhoGEF/RhoA signaling cascade to affect HIV-1 replication (Fig. 6). Further elucidation of the interaction among the specific GPCR, Gα13, and p115-RhoGEF will provide valuable information about the mechanism of possible GPCR-mediated RhoA activation and its effect on HIV-1 replication.

FIGURE 6.

FIGURE 6

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The RhoA activation pathway and HIV-1 replication. The putative GPCR linking Gα13 and the RhoA-signaling pathway may trigger a cascade of events to modulate HIV-1 replication, actin cytoskeletal organization (actin fiber), transcription activation (SRF), cell survival, and growth. Both the F39L and E40W mutants are defective in interacting with ROK and in promoting transformation of NIH3T3 cells (21). The E40W mutant is also defective in SRF activation (X). The F39L mutant is only partially defective in SRF activation (21, 22). The Y effector pathway is 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 inhibitory activity is defective in the F39L mutant, but functional in the E40W mutant. A novel effector (Z) pathway is proposed to mediate the HIV-1 inhibitory function.

The transmembrane glycoprotein (TM or gp41) of HIV-1 contains a long cytoplasmic domain (gp41C). Its function is not clear but has been implicated in regulating HIV-1 replication and cytopathogenicity (36). We recently demonstrated that HIV-1 gp41C 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 to impaired HIV-1 replication in various T cell lines (37). Thus, it is possible that one of the functions of HIV-1 gp41C is to counteract the inhibitory activity of p115/RhoA to enhance viral replication. However, the provirus bearing the mutations in gp41C produced WT levels of HIV virions when transfected in 293T cells (37), suggesting that lack of gp41C interaction (and inhibition) with p115 had no effect on HIV gene expression. Therefore, the inhibitory effect of gp41C on p115-mediated RhoA activation may not play a significant role in HIV-1 production in transfected 293T cells. We are currently investigating the mechanism of the impaired replication of the HIV mutants in T cell lines and in the SCID-hu Thy/Liv mouse.

The RhoA activity in inhibiting HIV-1 replication is distinct from its activities in cell transformation, SRF activation, and actin stress fiber formation. As proposed previously (21), the RhoA effector ROK is involved in cell transformation and partly in SRF activation and actin stress fiber formation (Fig. 6). Both the F39L and the E40W mutants have lost their interaction with ROK (21). An additional effector (X, defective in the E40W mutant) is involved in mediating SRF activation. A distinct effector (Y) is also proposed to mediate actin stress fiber formation (21, 22). In this report, a novel RhoA effector activity (Z) involved in the inhibition of HIV-1 replication is proposed. Mutant F39L is defective in the putative interaction (or activation) with Z, which will be useful to define the novel RhoA effector (Z).

Acknowledgments

We thank Drs. R. Swanstrom, Jenny Ting, Ian Whitehead, and Keith Burridge for discussion and critically reviewing this manuscript. We also thank the NIH AIDS Research and Reference Reagent Program for providing pNL4.Luc.R−E− (N. Landau), MAGI cells (M. Emerman) and T4-pMV7 (R. Axel), and the University of North Carolina, Capel Hill/Lineberger Comprehensive Cancer Center nucleic acid and DNA sequencing, flow cytometry, and tissue culture core facilities.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by a grant from the March of Dimes Basil O’Connor Scholar Award (to L.S.), and by National Institutes of Health Grants AI41356 (to L.S.) and CA63071 (to C.J.D.).

3
Abbreviations used in this paper:
SRF
serum response factor
GEF
guanine nucleotide exchange factor
RT
reverse transcriptase
ROK
Rho kinase
GPCR
G protein-coupled receptor
GAP
GTPase-activating protein
WT
wild type
CA
constitutively active
DH
Dbl homology
PH
pleckstrin homology

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