The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, L.
Right arrow Articles by Su, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L.
Right arrow Articles by Su, L.
The Journal of Immunology, 2000, 164: 5369-5374.
Copyright © 2000 by The American Association of Immunologists

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

Liping Wang*,{ddagger}, Hangchun Zhang{ddagger},§, Patricia A. Solski{dagger},{ddagger}, Matthew J. Hart, Channing J. Der{dagger},{ddagger} and Lishan Su2,*,{ddagger}

Departments of * Microbiology and Immunology and {dagger} Pharmacology, {ddagger} Lineberger Comprehensive Cancer Center, School of Medicine, and § Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC 27599; and Onyx Pharmaceuticals, Richmond, CA 94806


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{alpha}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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Rho family of small GTPases (RhoA, Rac1, and Cdc42) regulates a variety of important cell signaling and growth control pathways (1, 2, 3). In response to extracellular stimulation, activated Rho GTPases are involved in actin cytoskeletal reorganization (4, 5, 6), activation of transcription factors such as serum response factor (SRF)3 (7) or NF-{kappa}B (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, 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), rhotekin (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-{kappa}B (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, G{alpha}13, 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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, G{alpha}13, 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 ß-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 ß-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go, A–C). Both virion-associated p24 Ag (Fig. 1GoA) and RT activity (Fig. 1GoB) and infectious unit (Fig. 1GoC) 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. 1GoD). 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.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 1. 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, 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. 2Go, 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 G{alpha}13 (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. 2GoB). This control also showed that transfection efficiency was not affected by p115 and/or HIV-1.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. 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 G{alpha}13). 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-RhoGEF 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 G{alpha}13, which specifically activates p115-RhoGEF activity (15), led to inhibition of HIV-1 replication (Fig. 3GoA). Thus, activation of endogenous p115-RhoGEF by its upstream G protein also leads to inhibition of HIV-1 replication.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 3. Activation of endogenous p115-RhoGEF and RhoA by G{alpha}13 leads to HIV-1 inhibition. A, WT G{alpha}13 or CA G{alpha}13 was cotransfected with pNL4-3 and HIV-1 replication (RT x 103 cpm/ml) was measured. The experiments were performed three times with similar results. Error bars represent SDs. G{alpha}13WT, G{alpha}13CA, and vector indicate samples transfected with WT G{alpha}13, CA G{alpha}13, and pcDNA3 vector, respectively. B, RhoA (19N) counteracted the HIV-1 inhibitory activity of G{alpha}13. pNL4-3 and G{alpha}13CA were cotransfected with the dominant negative RhoA mutant RhoA (19N). HIV-1 replication (RT x 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 G{alpha}13 on HIV-1 replication (Fig. 3GoB). 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. 3GoC). 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. 4GoA). 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. 4GoA). 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.



View larger version (33K):
[in this window]
[in a new window]
  		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 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. 4GoB). 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. 5GoA). All RhoA mutant genes expressed similar levels of RhoA proteins (Fig. 5GoB and data not shown).



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5. 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. 5GoC and Table IGo). Although it has impaired activity in transformation of NIH3T3 cells, SRF activation, and actin stress fiber formation (Ref. 21 ; Table IGo and data not shown), the RhoA E40W mutant showed efficient inhibition of HIV-1 replication (Fig. 5GoC). 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. 5GoC). 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.


View this table:
[in this window]
[in a new window]
  		Table I. RhoA functions and effector loop mutants

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 (G{alpha}13) 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-{kappa}B (8). This hypothesis agrees with the finding that RhoA inhibited HIV-1 gene expression (Fig. 4Go). However, activation of NF-{kappa}B 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.

G{alpha}13 has recently been identified as an upstream activator of p115-RhoGEF (48). Its possible activation by putative G protein-coupled receptor (GPCRs, including the HIV-1 coreceptors; Ref. 5 2) suggests that extracellular factors (e.g., chemokines and HIV-1 env proteins) may modulate the G{alpha}13/p115-RhoGEF/RhoA signaling cascade to affect HIV-1 replication (Fig. 6Go). Further elucidation of the interaction among the specific GPCR, G{alpha}13, and p115-RhoGEF will provide valuable information about the mechanism of possible GPCR-mediated RhoA activation and its effect on HIV-1 replication.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 6. The RhoA activation pathway and HIV-1 replication. The putative GPCR linking G{alpha}13 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. 6Go). 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
 
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.). Back

2 Address correspondence and reprint requests to Dr. Lishan Su, Department of Microbiology and Immunology, 22-048 Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599. Back

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. Back

Received for publication October 19, 1999. Accepted for publication March 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hall, A.. 1994. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10:31.
  2. Hall, A.. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509.[Abstract/Free Full Text]
  3. Van Aelst, L., C. D’Souza-Schorey. 1997. Rho GTPases and signaling networks. Genes Dev. 11:2295.[Free Full Text]
  4. Ridley, A. J., A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389.[Medline]
  5. Ridley, A. J., H. F. Paterson, C. L. Johnston, D. Diekmann, A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401.[Medline]
  6. Nobes, C. D., A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53.[Medline]
  7. Hill, C. S., J. Wynne, R. Treisman. 1995. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81:1159.[Medline]
  8. Perona, R., S. Montaner, L. Saniger, I. Sanchez-Perez, R. Bravo, J. C. Lacal. 1997. Activation of the nuclear factor-{kappa}B by Rho, CDC42, and Rac-1 proteins. Genes Dev. 11:463.[Abstract/Free Full Text]
  9. Olson, M. F., A. Ashworth, A. Hall. 1995. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269:1270.[Abstract/Free Full Text]
  10. Khosravi-Far, R., P. A. Solski, G. J. Clark, M. S. Kinch, C. J. Der. 1995. Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol. Cell. Biol. 15:6443.[Abstract]
  11. Whitehead, I. P., S. Campbell, K. L. Rossman, C. J. Der. 1997. Dbl family proteins. Biochim. Biophys. Acta 1332:11.
  12. Marshall, C. J.. 1996. Ras effectors. Curr. Opin. Cell Biol. 8:197.[Medline]
  13. Leung, T., E. Manser, L. Tan, L. Lim. 1995. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270:29051.[Abstract/Free Full Text]
  14. Leung, T., X. Q. Chen, E. Manser, L. Lim. 1996. The p160 RhoA-binding kinase ROK {alpha} is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16:5313.[Abstract]
  15. Chihara, K., M. Amano, N. Nakamura, T. Yano, M. Shibata, T. Tokui, H. Ichikawa, R. Ikebe, M. Ikebe, K. Kaibuchi. 1997. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase. J. Biol. Chem. 272:25121.[Abstract/Free Full Text]
  16. Amano, M., H. Mukai, Y. Ono, K. Chihara, T. Matsui, Y. Hamajima, K. Okawa, A. Iwamatsu, K. Kaibuchi. 1996. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N. Science 271:648.[Abstract]
  17. Watanabe, G., Y. Saito, P. Madaule, T. Ishizaki, K. Fujisawa, N. Morii, H. Mukai, Y. Ono, A. Kakizuka, S. Narumiya. 1996. Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho. Science 271:645.[Abstract]
  18. Reid, T., T. Furuyashiki, T. Ishizaki, G. Watanabe, N. Watanabe, K. Fujisawa, N. Morii, P. Madaule, S. Narumiya. 1996. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J. Biol. Chem. 271:13556.[Abstract/Free Full Text]
  19. Hotta, K., K. Tanaka, A. Mino, H. Kohno, Y. Takai. 1996. Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor. Biochem. Biophys. Res. Commun. 225:69.[Medline]
  20. Madaule, P., T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, S. Narumiya. 1995. A novel partner for the GTP-bound forms of rho and rac. FEBS Lett. 377:243.[Medline]
  21. Sahai, E., A. S. Alberts, R. Treisman. 1998. RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 17:1350.[Medline]
  22. Zohar, M., H. Teramoto, B. Z. Katz, K. M. Yamada, J. S. Gutkind. 1998. Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation. Oncogene 17:991.[Medline]
  23. Reif, K., D. Cantrell. 1998. Networking Rho family GTPases in lymphocytes. Immunity 8:395.[Medline]
  24. Tousto, L., F. Michel, O. Acuto. 1996. p95Vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells. J. Exp. Med. 184:1161.[Abstract/Free Full Text]
  25. Stowers, L., D. Yelon, L. J. Berg, J. Chant. 1995. Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42. Proc. Natl. Acad. Sci. USA 92:5027.[Abstract/Free Full Text]
  26. Gallego, M. D., M. Santamaria, J. Pena, I. J. Molina. 1997. Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to Cd3-mediated stimulation. Blood 90:3089.[Abstract/Free Full Text]
  27. Woodside, D. G., D. K. Wooten, B. W. McIntyre. 1998. Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation. J. Exp. Med. 188:1211.[Abstract/Free Full Text]
  28. Galandrini, R., S. W. Henning, D. A. Cantrell. 1997. Different functions of the GTPase Rho in prothymocytes and late pre-T cells. Immunity 7:163.[Medline]
  29. Fauci, A. S.. 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384:529.[Medline]
  30. Zack, J. A., S. J. Arrigo, S. R. Weitsman, A. S. Go, A. Haislip, I. S. Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61:213.[Medline]
  31. Korin, Y. D., J. A. Zack. 1998. Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J. Virol. 72:3161.[Abstract/Free Full Text]
  32. Nabel, G., D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. [Published erratum appears in 1990 Nature 344:178.]. Nature 326:711.[Medline]
  33. Kinoshita, S., L. Su, M. Amano, L. A. Timmerman, H. Kaneshima, G. P. Nolan. 1997. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity 6:235.[Medline]
  34. Kinoshita, S., B. K. Chen, H. Kaneshima, G. P. Nolan. 1998. Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells. Cell 95:595.[Medline]
  35. Henning, S. W., R. Galandrini, A. Hall, D. A. Cantrell. 1997. The GTPase Rho has a critical regulatory role in thymus development. EMBO J. 16:2397.[Medline]
  36. Hunter, E., R. Swanstrom. 1990. Retrovirus envelope glycoproteins. Curr. Top. Microbiol. Immunol. 157:187.[Medline]
  37. Zhang, H., L. Wang, I. Whitehead, K. Duus, S. Kao, B. Liu, M. Hart, K. Burridge, C. Der, L. Su. 1999. Functional interaction between the cytoplasmic leucine zipper domain of HIV-1 TM gp41 and p115 RhoGEF. Curr. Biol. 9:1271.[Medline]
  38. Hart, M. J., S. Sharma, N. el Masry, R. G. Qiu, P. McCabe, P. Polakis, G. Bollag. 1996. Identification of a novel guanine nucleotide exchange factor for the Rho GTPase. J. Biol. Chem. 271:25452.[Abstract/Free Full Text]
  39. Whitehead, I. P., R. Khosravi-Far, H. Kirk, G. Trigo-Gonzalez, C. J. Der, R. Kay. 1996. Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors. J. Biol. Chem. 271:18643.[Abstract/Free Full Text]
  40. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284.[Abstract/Free Full Text]
  41. He, J., N. R. Landau. 1995. Use of a novel human immunodeficiency virus type 1 reporter virus expressing human placental alkaline phosphatase to detect an alternative viral receptor. J. Virol. 69:4587.[Abstract]
  42. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333.[Medline]
  43. Kimpton, J., M. Emerman. 1992. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated ß-galactosidase gene. J. Virol. 66:2232.[Abstract/Free Full Text]
  44. Su, L., H. Kaneshima, M. Bonyhadi, S. Salimi, D. Kraft, L. Rabin, J. M. McCune. 1995. HIV-1 induced thymocyte depletion is associated with indirect cytopathicity and infection of progenitor cells in vivo. Immunity 2:25.[Medline]
  45. Whitehead, I. P., K. Abe, J. L. Gorski, C. J. Der. 1998. CDC42 and FGD1 cause distinct signaling and transforming activities. Mol. Cell. Biol. 18:4689.[Abstract/Free Full Text]
  46. Hart, M. J., A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, Y. Zheng. 1994. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J. Biol. Chem. 269:62.[Abstract/Free Full Text]
  47. Olson, M. F., P. Sterpetti, K. Nagata, D. Toksoz, A. Hall. 1997. Distinct roles for Dh and Ph domains in the Lbc oncogene. Oncogene 15:2827.[Medline]
  48. Hart, M. J., X. Jiang, T. Kozasa, W. Roscoe, W. D. Singer, A. G. Gilman, P. C. Sternweis, G. Bollag. 1998. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G{alpha}13. Science 280:2112.[Abstract/Free Full Text]
  49. Mao, J., H. Yuan, W. Xie, D. Wu. 1998. Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein {alpha} subunit G{alpha}13. Proc. Natl. Acad. Sci. USA 95:12973.[Abstract/Free Full Text]
  50. Aasheim, H.-C., F. Pedeutour, E. B. Smeland. 1997. Characterization, expression and chromosomal localization of a human gene homologous to the mouse Lsc oncogene, with strongest expression in hematopoietic tissues. Oncogene 14:1747.[Medline]
  51. Goh, W. C., M. E. Rogel, C. M. Kinsey, S. F. Michael, P. N. Fultz, M. A. Nowak, B. H. Hahn, M. Emerman. 1998. HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nat. Med. 4:65.[Medline]
  52. Feng, Y., C. Broder, P. Kennedy, E. Berger. 1996. HIV-1 entry co-factor: functional cDNA cloning of a seven-transmembrane G-protein coupled receptor. Science 272:872.[Abstract]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
W. S. Helms, J. L. Jeffrey, D. A. Holmes, M. B. Townsend, N. A. Clipstone, and L. Su
Modulation of NFAT-dependent gene expression by the RhoA signaling pathway in T cells
J. Leukoc. Biol., August 1, 2007; 82(2): 361 - 369.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
V. McNeil Coffield, W. S. Helms, Q. Jiang, and L. Su
G{alpha}13 Mediates a Signal That Is Essential for Proliferation and Survival of Thymocyte Progenitors
J. Exp. Med., November 15, 2004; 200(10): 1315 - 1324.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
G. del Real, S. Jimenez-Baranda, E. Mira, R. A. Lacalle, P. Lucas, C. Gomez-Mouton, M. Alegret, J. M. Pena, M. Rodriguez-Zapata, M. Alvarez-Mon, et al.
Statins Inhibit HIV-1 Infection by Down-regulating Rho Activity
J. Exp. Med., August 16, 2004; 200(4): 541 - 547.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. E. Pontow, N. V. Heyden, S. Wei, and L. Ratner
Actin Cytoskeletal Reorganizations and Coreceptor-Mediated Activation of Rac during Human Immunodeficiency Virus-Induced Cell Fusion
J. Virol., July 1, 2004; 78(13): 7138 - 7147.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
I. Nobiron, D. R. O'Reilly, and J. A. Olszewski
Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda cells: a global analysis of host gene regulation during infection, using a differential display approach
J. Gen. Virol., November 1, 2003; 84(11): 3029 - 3039.
[Abstract] [Full Text] [PDF]

Home page
 Cell Growth Differ.Home page
P. A. Solski, W. Helms, P. J. Keely, L. Su, and C. J. Der
RhoA Biological Activity Is Dependent on Prenylation but Independent of Specific Isoprenoid Modification
Cell Growth Differ., August 1, 2002; 13(8): 363 - 373.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, L.
Right arrow Articles by Su, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L.
Right arrow Articles by Su, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS