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HIV relies on neddylation for ubiquitin ligase-mediated functions.

Nekorchuk MD, Sharifi HJ, Furuya AK, Jellinger R, de Noronha CM - Retrovirology (2013)

Bottom Line: Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G.It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1.This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, 43 New Scotland Avenue, Albany, NY 12208, USA. deNoroC@mail.amc.edu.

ABSTRACT

Background: HIV and SIV defeat antiviral proteins by usurping Cullin-RING E3 ubiquitin ligases (CRLs) and likely influence other cellular processes through these as well. HIV-2 viral protein X (Vpx) engages the cullin4-containing CRL4 complex to deplete the antiviral protein SAMHD1. Vif expressed by HIV-1 and HIV-2 taps a cullin5 ubiquitin ligase complex to mark the antiviral protein APOBEC3G for destruction. Viral Protein R of HIV-1 (Vpr) assembles with the CRL4 ubiquitin ligase complex to deplete uracil-N-glycosylase2 (UNG2). Covalent attachment of the ubiquitin-like protein side-chain NEDD8 functionally activates cullins which are common to all of these processes.

Results: The requirement for neddylation in HIV-1 and HIV-2 infectivity was tested in the presence of APOBEC3G and SAMHD1 respectively. Further the need for neddylation in HIV-1 Vpr-mediated depletion of UNG2 was probed. Treatment with MLN4924, an adenosine sulfamate analog which hinders the NEDD8 activating enzyme NAE1, blocked neddylation of cullin4A (CUL4A). The inhibitor hindered HIV-1 infection in the presence of APOBEC3G, even when Vif was expressed, and it stopped HIV-2 infection in the presence of SAMHD1 and Vpx. Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G. It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1.

Conclusions: Neddylation plays an important role in HIV-1 and HIV-2 infection. This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

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Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(−)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).
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Figure 4: Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(−)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).

Mentions: Infection of HEK293T cells with Vpr-expressing HIV-1 caused a dramatic decrease in endogenous UNG2 levels (Figure 4A, endog.). Surprisingly, application of MLN4924 after infection barely blocked UNG2 depletion in these cultures. The observation that endogenous UNG2 depletion was not blocked more completely despite the robust loss of neddylated CUL4A species, led us to hypothesize that Vpr may be restricting UNG2 expression through both NEDD8-dependent and -independent mechanisms. The latter could be mediated through a cullin-independent ubiquitin ligase that is not impacted by a neddylation block, or by another mechanism altogether. Langevin et al., for example, showed that Vpr expression hinders UNG2 production at the level of transcription[26]. Indeed, when we expressed UNG2, tagged with dual HA epitope tags (UNG2–2HA) from an expression vector using a CMV-IE promoter, UNG2–2HA levels were maintained upon infection with HIV-1 in the presence of MLN4924 (Figure 4A, exog.).


HIV relies on neddylation for ubiquitin ligase-mediated functions.

Nekorchuk MD, Sharifi HJ, Furuya AK, Jellinger R, de Noronha CM - Retrovirology (2013)

Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(−)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).
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Figure 4: Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(−)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).
Mentions: Infection of HEK293T cells with Vpr-expressing HIV-1 caused a dramatic decrease in endogenous UNG2 levels (Figure 4A, endog.). Surprisingly, application of MLN4924 after infection barely blocked UNG2 depletion in these cultures. The observation that endogenous UNG2 depletion was not blocked more completely despite the robust loss of neddylated CUL4A species, led us to hypothesize that Vpr may be restricting UNG2 expression through both NEDD8-dependent and -independent mechanisms. The latter could be mediated through a cullin-independent ubiquitin ligase that is not impacted by a neddylation block, or by another mechanism altogether. Langevin et al., for example, showed that Vpr expression hinders UNG2 production at the level of transcription[26]. Indeed, when we expressed UNG2, tagged with dual HA epitope tags (UNG2–2HA) from an expression vector using a CMV-IE promoter, UNG2–2HA levels were maintained upon infection with HIV-1 in the presence of MLN4924 (Figure 4A, exog.).

Bottom Line: Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G.It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1.This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, 43 New Scotland Avenue, Albany, NY 12208, USA. deNoroC@mail.amc.edu.

ABSTRACT

Background: HIV and SIV defeat antiviral proteins by usurping Cullin-RING E3 ubiquitin ligases (CRLs) and likely influence other cellular processes through these as well. HIV-2 viral protein X (Vpx) engages the cullin4-containing CRL4 complex to deplete the antiviral protein SAMHD1. Vif expressed by HIV-1 and HIV-2 taps a cullin5 ubiquitin ligase complex to mark the antiviral protein APOBEC3G for destruction. Viral Protein R of HIV-1 (Vpr) assembles with the CRL4 ubiquitin ligase complex to deplete uracil-N-glycosylase2 (UNG2). Covalent attachment of the ubiquitin-like protein side-chain NEDD8 functionally activates cullins which are common to all of these processes.

Results: The requirement for neddylation in HIV-1 and HIV-2 infectivity was tested in the presence of APOBEC3G and SAMHD1 respectively. Further the need for neddylation in HIV-1 Vpr-mediated depletion of UNG2 was probed. Treatment with MLN4924, an adenosine sulfamate analog which hinders the NEDD8 activating enzyme NAE1, blocked neddylation of cullin4A (CUL4A). The inhibitor hindered HIV-1 infection in the presence of APOBEC3G, even when Vif was expressed, and it stopped HIV-2 infection in the presence of SAMHD1 and Vpx. Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G. It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1.

Conclusions: Neddylation plays an important role in HIV-1 and HIV-2 infection. This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

Show MeSH
Related in: MedlinePlus