The recruitment of PML to HIV-1 PICs could promote its association with PML-interacting proteins such as the his- tone acetyltransferase (HAT) CBP/p300, or with other transcription factors. Similarly, the binding of INI1 to the HIV-1 PICs may trigger the recruitment of the SWI/SNF complex to the PIC, possibly targeting HIV integration to actively transcribed regions of the genome or facilitating subsequent expression of the provirus. To probe these issues, we investigated the potential roles of INI1 and PML in HIV-1 integration and transcription.
The Tat protein is a potent activator of humanimmunodeficiencyvirustype1transcription. Tat has been shown to act by increasing both transcription initiation and elongation, but a detailed understanding of its interaction with the transcriptional machinery is lacking. With the aim of isolating cellular proteins that interact with Tat and play a role in transactivation, we have reexamined its function in a cell-free transcription assay. Monitoring the appearance of transactivation after addition of purified Tat at intervals to the reaction mix revealed a lag of approximately 10 min before Tat is able to effect transactivation. Incubation of Tat in nuclear or cytoplasmic extracts of human cells was sufficient to eliminate the lag, but nuclear extract from a rodent cell line was inactive. The accelerating effect of the human cell extract could be abrogated by dilution, heat inactivation, or chromatographic depletion. We infer that Tat is potentiated for transactivation through interaction with a protein factor(s) that is specific to human cells.
We observed striking specificity of the dominant negative proteins, as replication was inhibited only in viruses driven by a noncognate RNA-protein interaction. Expression of T-U2AF65, lacking a TAR RBD, markedly suppressed rep- lication of both viruses compared to the Tat AD or U2AF65 controls, with no p24 antigen detectable until 18 to 20 days after infection. Expression of Tat-U2AF65 or T-BIV- U2AF65 inhibited replication of the noncognate virus to an extent similar to that of T-U2AF65 and only slightly inhib- ited the cognate virus (Fig. 8A and B). Interestingly, expres- sion of Tat or T-BIV activators accelerated replication of the cognate viruses but not the noncognate viruses, suggest- ing that Tat levels in these viruses may be limiting. The strong levels of inhibition are notable, given that expression levels of the inhibitors are low in these cell lines, as judged by Western blot analysis of samples immunoprecipitated
Therefore, it was of interest to determine whether these mu- tations disrupted RT-IN interaction. We performed GST-IN pull-down experiments using each of these mutant IN proteins.
As illustrated in Fig. 7, our results show that none of the mutations we tested has any effect on the ability of GST-IN to pull down RT from crude lysates. These results suggest that the defect in reverse transcription exhibited by mutant viruses FIG. 3. Mapping the IN-binding domain on HIV-1 RT. In experiments similar to those shown in Fig. 2, G beads bound to GST or GST-IN were incubated with bacterial lysates containing various truncation mutants of RT and washed, and the bound proteins were resolved on duplicate SDS-PAGE gels. The proteins on one gel were transferred to nitrocellulose and subjected to immunostaining with ␣-RT antibodies, and the second gel was stained with Coomassie blue to ensure equal input of the bait proteins. (A) Pull-down experiments to map the IN-binding domains on RT p66. Lanes 1, 5, 7, 9, 11, and 13 contained bound proteins from a control incubation of GST bound to G beads, and lanes 2 to 4, 6, 8, 10, 12, and 14 to 17 contained proteins bound to GST-IN bound to G beads. RT mutants were added to the lanes as follows: lane 2, p66; lane 3, TCR; lane 4, Conn-R; lanes 5 and 6, C*R; lanes 7 and 8, R; lanes 9 and 10, p66; lanes 11 and 12, T; lanes 13 and 14, p66; lane 15, p51; lane 16, FPT; and lane 17, FP. The corresponding lanes on the bottom show stained protein indicating the input bait protein levels. The differences in p66 intensities in lanes 2 and 14 are due to different antibodies used for the Western blots. (B) A schematic summarizing results obtained in the experiment shown in panel A. The horizontal bar at the top represents full-length RT p66, with various subdomains and their boundaries indicated by amino acid residue numbers. The RT truncations used in the pull-down experiment, their boundaries, and their abilities to bind IN are indicated. Below, the proposed domains of IN interaction are indicated. ⫹, able to bind; ⫺, not able to bind.
Received 23 February 2007/Accepted 2 May 2007
The humanimmunodeficiencyvirustype1 (HIV-1) RNA genome contains a terminal repeat (R) region that encodes the transacting responsive (TAR) hairpin, which is essential for Tat-mediated activation of gene expression. TAR has also been implicated in several other processes during viral replication, including translation, dimerization, packaging, and reverse transcription. However, most studies in which replication of TAR-mutated viruses was analyzed were complicated by the dominant negative effect of the mutations on transcription. We therefore used an HIV-1 variant that does not require TAR for transcription to reinvestigate the role of TAR in HIV-1 replication. We demonstrate that this virus can replicate efficiently upon complete deletion of TAR. Furthermore, evolution of a TAR-deleted variant in long-term cultures indicates that HIV-1 requires a stable stem-loop structure at the start of the viral transcripts in which the 5 ⴕ-terminal nucleotides are base paired. This prerequisite for efficient replication can be fulfilled by the TAR hairpin but also by unrelated stem-loop structures. We therefore conclude that TAR has no essential function in HIV-1 replication other than to accommodate Tat-mediated activation of transcription.
FRANCESCA DEMARCHI, FABRIZIO D ’ADDA DI FAGAGNA, ARTURO FALASCHI, AND MAURO GIACCA*
International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy Received 24 January 1996/Accepted 26 March 1996
A recombinant Tat protein was used to investigate the molecular mechanisms of transcriptional activation of the humanimmunodeficiencyvirustype1 long terminal repeat (LTR). Liposome-mediated delivery of this protein to responsive cells results in dose-dependent LTR activation. As evaluated by mRNA quantitation with competitive PCR, the activation response is rapid and transient, peaking at 5 h after the beginning of Tat treatment. In vivo footprinting experiments at the LTR showed that transcriptional activation is concomitant with a modification of the protein-DNA interaction pattern at the downstream k B site of the enhancer and at the adjacent Sp1 boxes. The effects of Tat on the enhancer are mediated by Tat-induced nuclear translocation of NF- k B, which parallels the kinetics of transcriptional activation. This induction results from degradation of the inhibitor I k B- a , is blocked under antioxidant conditions and by a protease inhibitor, and occurs as a rapid response in different cell types. The functional response to Tat is impaired upon cell treatment with a k B site decoy or with sodium salicylate, an inhibitor of NF- k B activation. These results show that NF- k B activation by Tat is important for LTR transcriptional activation. Furthermore, they suggest that some of the pleiotropic effects of Tat on cellular functions can be mediated by induction of NF- k B.
These results were consistent for four to six independent virus stocks and suggest that the ability of tat to efficiently initiate HIV-1 reverse transcription is largely dependent on an intact Tat amino terminus. There is an additional requirement for the basic domain of Tat to fully complement HIV-1 reverse transcription. Surprisingly, amino acid residues within the Tat cysteine and core domains that are necessary for tat-mediated activation of HIV-1 gene expression are not required for tat stimulation of HIV-1 reverse transcription. We observed sim- ilar patterns of reverse transcription complementation in PB- MCs infected with virus stocks produced by transient expres- sion of these genes into 293 Dtat cells, although there was some variability in the overall degree of complementation (data not shown).
Significant amino acid variations have been observed among the clade-specific Tatproteins. For the present study, we examined clade-specific interactions between Tat, transactivation-responsive (TAR) element, and P-TEFb proteins and how these interactions may modulate the efficiency of HIV-1transcription. Clade-specific Tatproteins significantly modified viral gene expression. Tatproteins derived from HIV-1 clades C and E were strong transactivators of long terminal repeat (LTR) activity; Tat E also had a longer half-life than the other Tatproteins and interacted more efficiently with the stem-loop TAR element. Chimeric Tatproteins harboring the Tat E activation domain were strong transactivators of LTR expression. While Tat B, C, and E were able to rescue a Tat-defective HIV-1 proviral clone, Tat E was significantly more efficient at rescue than Tat C, possibly due to the relative stability of the Tat protein. Swapping the activation domains of Tat B, C, and E identified the cyclin T1 association domain as a critical determinant of the transactivation efficiency and of Tat-defective HIV-1 provirus rescue.
Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan 2
Received 23 November 2005/Accepted 27 March 2006
Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the humanimmunodeficiencyvirustype1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.
In this work, we make three salient points. First, we observed a good correlation between inactive Tat mutants that compete with wild-typeTat for Sp1 contact and ones that repress HIV-1 replication in cultured T cells (Fig. 1 and 10). A simple corol- lary of such finding, which does not exclude others, is that wild-typeTat-Sp1 contact is physiologically important for pro- ductive HIV-1 infection. Relevant to this idea is the in situ observation that within intact primate cell nuclei, a population of Tat and Sp1 proteins colocalizes. Second, we observed a contribution of phosphorylated Sp1 for Tat transactivation ac- tivity. Although performed on a subfragment of transcription- ally active Sp1, the experiments in Fig. 8 and 9 indicate that within a defined context, serine 131 phosphorylation is impor- tant for supporting Tat-activated transcription from the HIV-1 promoter. There are 78 serines in full-length Sp1. Presumably, phosphorylation at other residues can differentially affect other functional activities. Third, we propose a regulatory loop in which Tat serves to influence the phosphorylation state of Sp1.
previously reported that reverse transcription is at least partly dependent on the Tat basic domain, we next asked whether mutations in this domain affect cleavage by PR. We also asked whether amino acid residues that flank the basic domain, mu- tations in which do not abolish transactivation but have been reported to delay the replication cycle in T cells, also contrib- ute to reverse transcription and/or cleavage by PR. We found that Tat amino acids 49 to 52 did contribute to the role played by Tat in reverse transcription and that there was a striking correlation between the efficiency with which PR cleaved Tatproteins with mutations in the basic domain or flanking se- quence and the ability of each mutant to enhance reverse transcription. The sole exception to this latter observation was that tyrosine 47 was required for full Tat function in reverse transcription in both cell infection and NERT but did not af- fect PR cleavage of Tat. Mutation of Y47 to aspartic acid (which preserves overlapping reading frames) greatly reduced virus replication but did not substantially affect transcription. On the basis of these results, we hypothesize that Tat is a reverse transcription accessory factor, that this activity is regulated by PR, and that the cleaved form of Tat is a virion protein which requires Y47 for its function in reverse transcription.
Isolated cytoplasmic PIC can insert the viral DNA ends in a concerted fashion into exogenously supplied target DNA sub- strates in vitro (3, 5, 6, 22, 33, 50).
Major advances have occurred in identifying target sites and understanding the basis for target site selectivity exhibited by humanimmunodeficiencyvirustype1 (HIV-1), murine leuke- mia virus (MLV), and avian sarcoma-leukemia virus in human chromosomes (25, 34, 36, 37, 51). HIV-1 predominantly inte- grates within genes, having a preference for those that are actively transcribing. MLV prefers integration into or near transcription start sites, while avian sarcoma-leukemia virus exhibits a slight preference for genes and no preference for transcription start sites. These results suggest that HIV-1 and MLV may interact with chromatin-associated factors and/or transcriptional cofactors that direct the PIC to transcription units in the case of HIV-1 and to or near transcriptional start
One interesting finding is that the truncated integrases IN1- 234 and IN50-234 showed a weak 3 9 -end joining activity when assayed by the sensitive PCR-based method; no 3 9 -end joining activity was detectable in the conventional in vitro assays. We also observed a weak 3 9 -end joining activity when we per- formed the same PCR assay with a D116N mutant, which contains an asparagine instead of the highly conserved aspartic acid at position 116 (data not shown). The weak 3 9 -end joining activity observed with the truncated integrases and the D116N mutant was not changed in the presence or absence of the N-terminal His tag (data not shown). The D116N mutant has been shown previously to be inactive in all known catalytic activities of integrase in the conventional assays (13, 26, 27, 49). Control experiments were carried out to confirm that the observed 3 9 -end joining activities of the truncated integrases and D116N mutant were not due to a contamination of the PCR (data not shown). The similarity among the mutant and wild-type integrases in the banding pattern on a sequencing gel further supports the view that the PCR-amplified products were not experimental artifacts and that the truncated inte- grases and D116N mutant indeed possess 39-end joining activ- ity. We think that this finding has important significance for in vivo experiments in which putatively integration-defective vi- ruses are studied. Several studies have shown that viruses con- taining a mutation at D-116 of integrase, although not able to replicate, are positive for an indicator cell assay that requires the expression of Tat protein (1, 14, 57). The results are gen- erally interpreted as evidence that unintegrated viral DNA may serve as a template for Tat expression. In light of the weak 3 9 -end joining activity of the D116N mutant, it is possible that viruses containing a D-116 mutation of integrase may be ca- pable of forming a low level of proviruses, which may in turn produce sufficient Tat protein for the indicator cell assay.
PRMT6 is a type I methyltransferase involved in regulating base excision repair (14), suppressing the HMGA1a architec- tural transcription factor (51), and controlling transcription by methylating histone H3 (22, 26). Arginine methylation by PRMT6 was recently shown to have a negative impact on the activities of HIV-1Tat, Rev, and nucleocapsid proteins (5, 27, 28). PRMT6 methylates Tat at arginine residues 52 and 53 and consequently disrupts the Tat-TAR-cyclin T1 complex re- quired for transactivation (58), presumably by inhibiting inter- actions between R52/R53 and the bulge region of TAR. It is therefore thought that PRMT6 is a negative regulator of Tat function, but the fate and functions, if any, of methylated Tat are unknown. Here we found that overexpression of PRMT6 led to a significant increase in Tat stability in a manner requir- ing PRMT6 catalytic activity. The increase in stability was due to prevention of Tat degradation by a proteasome. Our data raise the possibility that PRMT6 generates a distinct subset of stable Tat molecules able to perform functions other than Tat’s established role in HIV-1 gene expression.
Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between inte- gration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.
two proteins is supported by biochemical analyses in vitro (28, 50, 58). The specific RT-IN interaction is also confirmed by a pull-down assay with glutathione S-transferase or His tag, and the RT-binding site on IN is mapped to the C-terminal domain (27a), corroborating our results reported here. The finding that IN interacts with RT is consistent with the requirement for IN during reverse transcription. However, the question of whether the RT-IN interaction observed in vitro is biologically signifi- cant has not been addressed. Also, it has not been determined if the IN mutations that fail to support reverse transcription in vivo are incapable of interacting with RT in vitro. In this study, we showed that INs containing Cys substitutions at positions 56 and 65, which can support the initiation of reverse transcrip- tion, retain the ability to physically interact with RT. Con- versely, viruses harboring the C130S mutation have a complete absence of reverse-transcription product, and purified C130S- containing IN fails to interact with RT in vitro. Although the mechanism is still unknown, these results reveal a direct cor- relation between IN mutations that impair reverse transcrip- tion in vivo and the ability of the IN mutant to interact with RT in vitro. Taken together, the data suggest that the RT-IN interaction in vitro is biologically relevant and has an impor- tant function during viral replication. Experiments are under way to examine the abilities of other known reverse-transcrip- tion-defective IN mutants to coimmunoprecipitate with RT and to determine whether the physical interaction between RT and IN occurs in vivo.
TASK and Vpu proteins preferentially suppress unintegrated HIV-1 DNA
We have shown that TASK proteins and Vpu suppress viral transcription in cells transfected with HIV-1 pro- viral DNA. During these transient transfection assays, the HIV-1 provirus remains unintegrated. Therefore we sought to examine the effect of TASK and Vpu proteins on HIV-1transcription from integrated and unintegrated viral genomes. For this purpose we determined the effect of TASK and Vpu expression on HIV-1transcription in cells that had contained an integrated and an uninte- grated viral genome. We used the JLTRG cell line which is derived from the Jurkat cell line and has been stably transfected with an HIV LTR-GFP construct. The JLTRG cells were transfected with a Tat expression plasmid along with HIV-LTR-DsRed, TASK or Vpu expression vectors or empty vector control. In the absence of HIV- 1 infection or HIV-1Tat expression the cells exhibit no
In contrast to the C-terminally truncated integrase fused to LexA, the fusion protein containing the N-terminally truncated integrase is much worse at restoring integration to the inte- grase-mutated viral clone. This may be because the N terminus of integrase plays multiple roles during retroviral infection and complementation of these functions by the core-mutated inte- grase encoded by the virus is difficult. Viruses containing mu- tations in the conserved HHCC motif of this domain have abnormal morphology, suggesting that the N terminus of inte- grase may be involved in maturation of particles (16). In ad- dition, these viruses produce reduced levels of viral cDNA, implicating the N terminus of integrase as playing a role during reverse transcription (16, 42, 49, 68, 73). Integration may also be negatively affected because integrase may be prevented from multimerizing (43, 75) or recognizing the viral cDNA ends (62, 65). The C terminus of integrase, on the other hand, may play a role only during the integration step of the retro- viral life cycle, which would be more easily complemented by the core-mutated integrase. In vitro assays indicate that the C-terminal domain of integrase is involved in nonspecific- DNA binding (17, 35, 53, 58, 66, 70, 71), and replacing the C-terminal domain of HIV-1integrase with LexA, a DNA- binding protein, may enable this fusion protein to better re- store integration activity than the N-terminally truncated inte- grase-LexA can. Therefore, although both of these fusion proteins would be able to provide a catalytic motif to the core-mutated integrase encoded by the HXB 2 clone, the ability of the core-mutated integrase to restore functions provided by an N-terminally deleted integrase fused to LexA may be more difficult.
We found here that human EED, a Pc-G protein, can inter- act with HIV-1 IN both in vitro and in vivo in yeast. Using deletion mutagenesis and phage biopanning, we mapped the major EED-binding sites to the C-terminal domain of IN. In vitro, we observed an apparent positive effect of EED on IN- mediated integration reaction. We hypothesize that this effect was indirect: the interaction of EED with IN could promote the oligomerization of IN molecules, which would in turn favor the integration process. In situ analysis of EED and IN cellular localization was performed on HIV-1-infected human epithe- lial (HeLa CD4 ⫹ ) or lymphoid (MT4) cells by using IEM and differential immunogold labeling. We found that EED and IN colocalized within the nucleus of HIV-infected cells, a phe- nomenon that was mainly observed at early times p.i. (1.5 to 6 h). Triple-immunolabeling experiments showed that the MA protein, another viral protein partner of EED (54), was also detected in significantly frequent associations with both EED and IN proteins in the nucleus at early times p.i., suggesting the occurrence of ternary complexes involving EED, MA, and IN.