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TOX4 and NOVA1 proteins are partners of the LEDGF PWWP domain and affect HIV-1 replication.

Morchikh M, Naughtin M, Di Nunzio F, Xavier J, Charneau P, Jacob Y, Lavigne M - PLoS ONE (2013)

Bottom Line: Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches.These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA.Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Laboratoire Joliot-Curie, Centre National de la Recherche Scientifique, Lyon, France ; Institut Pasteur, Unité de Virologie Structurale, Centre National de la Recherche Scientifique, Unité de recherche associée, Paris, France ; Université Pierre et Marie Curie, Paris, France.

ABSTRACT
PWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.

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Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.
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pone-0081217-g006: Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.

Mentions: The LEDGF IBD-Integrase interaction is crucial for HIV replication and overexpression of this IBD in infected cells strongly inhibits this process by competing for the interaction with integrase [33], [35], [80]. The LEDGF PWWP domain is also important for HIV replication as it targets integrase to cellular chromatin [36], [38]. We wondered if an overexpression of the two identified PWWP partners could also affect the efficiency of replication. This question was addressed by infecting Hela CD4 CCR5 cells that transiently express Flag-TOX4 PIR, Flag-NOVA1 PIR or Flag-LEDGF IBD. The HIV-1 strain used for this study is pseudotyped for the VSV-G envelope and codes for the luciferase gene. As expected, in 3 independent experiments, we observed a significant reduction of viral infectivity in cells that transiently express the LEDGF IBD (2.2 fold effect in the experiment presented in Figure 6A). This effect is lower than the one previously observed in cells stably over-expressing GFP-IBD [33], [35]. We also observed a significant decrease of viral infectivity after a transient expression of NOVA1 or TOX4 PIRs (3.8 and 2.2 fold in the experiment presented in Figure 6A). We also tested the effect on viral replication of four other PIRs identified by the Y2H screen but not selected by PCA in 293T cells : BC0631, COP5, CNRIP1 and RLF. We observed similar levels of HIV infectivity in HeLa cells transiently expressing these proteins, while using the same conditions we detected a decrease in HIV infectivity in cells expressing the TOX4, NOVA1 or IBD constructs (Figure S4A). We also evaluated the level of expression of TOX4, NOVA1, IBD, BC0631, COP5, CNRIP1 and RLF by western blotting of the total cell extracts at the moment of virus challenge and we did not observe significant differences that could explain the effects observed on infectivity (Figure S4B).


TOX4 and NOVA1 proteins are partners of the LEDGF PWWP domain and affect HIV-1 replication.

Morchikh M, Naughtin M, Di Nunzio F, Xavier J, Charneau P, Jacob Y, Lavigne M - PLoS ONE (2013)

Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842248&req=5

pone-0081217-g006: Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.
Mentions: The LEDGF IBD-Integrase interaction is crucial for HIV replication and overexpression of this IBD in infected cells strongly inhibits this process by competing for the interaction with integrase [33], [35], [80]. The LEDGF PWWP domain is also important for HIV replication as it targets integrase to cellular chromatin [36], [38]. We wondered if an overexpression of the two identified PWWP partners could also affect the efficiency of replication. This question was addressed by infecting Hela CD4 CCR5 cells that transiently express Flag-TOX4 PIR, Flag-NOVA1 PIR or Flag-LEDGF IBD. The HIV-1 strain used for this study is pseudotyped for the VSV-G envelope and codes for the luciferase gene. As expected, in 3 independent experiments, we observed a significant reduction of viral infectivity in cells that transiently express the LEDGF IBD (2.2 fold effect in the experiment presented in Figure 6A). This effect is lower than the one previously observed in cells stably over-expressing GFP-IBD [33], [35]. We also observed a significant decrease of viral infectivity after a transient expression of NOVA1 or TOX4 PIRs (3.8 and 2.2 fold in the experiment presented in Figure 6A). We also tested the effect on viral replication of four other PIRs identified by the Y2H screen but not selected by PCA in 293T cells : BC0631, COP5, CNRIP1 and RLF. We observed similar levels of HIV infectivity in HeLa cells transiently expressing these proteins, while using the same conditions we detected a decrease in HIV infectivity in cells expressing the TOX4, NOVA1 or IBD constructs (Figure S4A). We also evaluated the level of expression of TOX4, NOVA1, IBD, BC0631, COP5, CNRIP1 and RLF by western blotting of the total cell extracts at the moment of virus challenge and we did not observe significant differences that could explain the effects observed on infectivity (Figure S4B).

Bottom Line: Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches.These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA.Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Laboratoire Joliot-Curie, Centre National de la Recherche Scientifique, Lyon, France ; Institut Pasteur, Unité de Virologie Structurale, Centre National de la Recherche Scientifique, Unité de recherche associée, Paris, France ; Université Pierre et Marie Curie, Paris, France.

ABSTRACT
PWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.

Show MeSH
Related in: MedlinePlus