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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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MLN4924 inhibits infection of macrophages by HIV-2. Primary human MDMs (A) or HEK293T cells (B) were pre-treated with 1 μM MLN4924 for three hours, and infected at an equivalent MOI with VSV-G-pseudotyped HIV-2 or HIV-2 with a frame-shift mutation in Vpx, Vpr or both as indicated. Panels C and D summarize data from multiple experiments and error bars show standard error. All viral constructs expressed GFP in place of Nef. GFP expression was detected using flow cytometry and used as an indicator of infection. The two-tailed Student’s t-test was used to determine whether differences between pairs of conditions are statistically significant. ns indicates comparisons where the differences were not significant at a level of 0.05.
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Figure 1: MLN4924 inhibits infection of macrophages by HIV-2. Primary human MDMs (A) or HEK293T cells (B) were pre-treated with 1 μM MLN4924 for three hours, and infected at an equivalent MOI with VSV-G-pseudotyped HIV-2 or HIV-2 with a frame-shift mutation in Vpx, Vpr or both as indicated. Panels C and D summarize data from multiple experiments and error bars show standard error. All viral constructs expressed GFP in place of Nef. GFP expression was detected using flow cytometry and used as an indicator of infection. The two-tailed Student’s t-test was used to determine whether differences between pairs of conditions are statistically significant. ns indicates comparisons where the differences were not significant at a level of 0.05.

Mentions: As expected from previously published data, we observed robust infection with the viruses expressing Vpx and comparatively modest infection with the viruses lacking the capacity to express Vpx (Figure 1A, c and g versus e and i, and C). Addition of the neddylation inhibitor reduced infection of Vpx-expressing viruses to levels comparable with those lacking the capacity to express Vpx (Figure 1A, c and d, g and h, and C). While infection levels with Vpx(−) virus were relatively low, they were further reduced by MLN4924-treatment (Figure 1A, e and f, i and j, and C). It’s possible that the additional reduction in Vpx(−) virus infection is due to another requirement for cullin-containing ubiquitin ligases or due to an off-target effect on MDMs at this dose. If the MLN4924-mediated block of infection is due to the anti-viral activity of SAMHD1, we hypothesized that an equal dose of this compound would not block HIV-2 infection in HEK293T cultures. SAMHD1 does not exhibit anti-viral activity in this cell-type regardless of whether Vpr or Vpx is absent (Figure 1B, c and g versus e and i, and D). Addition of MLN4924 did not alter the overall pattern of infection in any of the cultures (Figure 1B left column versus right column). Slightly fewer cells were observed to be infected in the presence of the drug but this did not correlate with the presence of Vpx. Of note, we will show in subsequent figures that MLN4924 is biologically active in HEK293T cells at the dose used here and that the dose of MLN4924 used in these experiments has no significant impact on cell health during the time-frame of the experiments (Additional file1: Figure S1).


HIV relies on neddylation for ubiquitin ligase-mediated functions.

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

MLN4924 inhibits infection of macrophages by HIV-2. Primary human MDMs (A) or HEK293T cells (B) were pre-treated with 1 μM MLN4924 for three hours, and infected at an equivalent MOI with VSV-G-pseudotyped HIV-2 or HIV-2 with a frame-shift mutation in Vpx, Vpr or both as indicated. Panels C and D summarize data from multiple experiments and error bars show standard error. All viral constructs expressed GFP in place of Nef. GFP expression was detected using flow cytometry and used as an indicator of infection. The two-tailed Student’s t-test was used to determine whether differences between pairs of conditions are statistically significant. ns indicates comparisons where the differences were not significant at a level of 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842660&req=5

Figure 1: MLN4924 inhibits infection of macrophages by HIV-2. Primary human MDMs (A) or HEK293T cells (B) were pre-treated with 1 μM MLN4924 for three hours, and infected at an equivalent MOI with VSV-G-pseudotyped HIV-2 or HIV-2 with a frame-shift mutation in Vpx, Vpr or both as indicated. Panels C and D summarize data from multiple experiments and error bars show standard error. All viral constructs expressed GFP in place of Nef. GFP expression was detected using flow cytometry and used as an indicator of infection. The two-tailed Student’s t-test was used to determine whether differences between pairs of conditions are statistically significant. ns indicates comparisons where the differences were not significant at a level of 0.05.
Mentions: As expected from previously published data, we observed robust infection with the viruses expressing Vpx and comparatively modest infection with the viruses lacking the capacity to express Vpx (Figure 1A, c and g versus e and i, and C). Addition of the neddylation inhibitor reduced infection of Vpx-expressing viruses to levels comparable with those lacking the capacity to express Vpx (Figure 1A, c and d, g and h, and C). While infection levels with Vpx(−) virus were relatively low, they were further reduced by MLN4924-treatment (Figure 1A, e and f, i and j, and C). It’s possible that the additional reduction in Vpx(−) virus infection is due to another requirement for cullin-containing ubiquitin ligases or due to an off-target effect on MDMs at this dose. If the MLN4924-mediated block of infection is due to the anti-viral activity of SAMHD1, we hypothesized that an equal dose of this compound would not block HIV-2 infection in HEK293T cultures. SAMHD1 does not exhibit anti-viral activity in this cell-type regardless of whether Vpr or Vpx is absent (Figure 1B, c and g versus e and i, and D). Addition of MLN4924 did not alter the overall pattern of infection in any of the cultures (Figure 1B left column versus right column). Slightly fewer cells were observed to be infected in the presence of the drug but this did not correlate with the presence of Vpx. Of note, we will show in subsequent figures that MLN4924 is biologically active in HEK293T cells at the dose used here and that the dose of MLN4924 used in these experiments has no significant impact on cell health during the time-frame of the experiments (Additional file1: Figure S1).

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