Limits...
HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2.

Wagner RN, Reed JC, Chanda SK - Retrovirology (2015)

Bottom Line: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR.Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB.These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA. rwagner@sbpdiscovery.org.

ABSTRACT

Background: HIV-1 protease (PR) is essential for viral infectivity as it cleaves Gag and Gag-Pol polyprotein precursors during viral maturation. Recent evidence suggests that cellular proteins can also be cleaved by PR, perhaps representing an important viral strategy to counter host defense mechanisms. Receptor-interacting protein kinase 1 (RIPK1) and RIPK2 belong to a family of serine/threonine kinases with conserved domain architecture and important functions in apoptosis, necrosis and innate immunity.

Results: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR. In RIPK1, we identified a putative PR cleavage site; a mutation at this site rendered RIPK1 resistant to PR cleavage. RIPK1 and RIPK2 were cleaved during HIV-1 infection of T cell lines or primary activated CD4(+) T cells. Interfering with the viral life cycle at different stages by the addition of specific inhibitors against RT, integrase, or PR, completely prevented RIPK1 and RIPK2 cleavage. Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB.

Conclusions: These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.

No MeSH data available.


Related in: MedlinePlus

Identification of the PR cleavage site in RIPK1. a Primary sequence of human RIPK1 (aa 449–476 according to NCBI Reference Sequence NP_003795.2) with PR cleavage site between residues 462 and 463. Indicated point mutations were introduced by Quikchange mutatgenesis. b HEK293T cells were transfected with expression plasmids encoding wild-type (wt) RIPK1, RIPK1 with a double-mutation in the PR cleavage site (RIPK1 PRres), or RIPK1 with a non-sense mutation in the PR cleavage site (RIPK1 ΔC), respectively, along with catalytically active HIV PR (5 ng/well) in the absence (−) or presence (+) of SQV (5 μM). Cell lysates were subjected to SDS-PAGE and immunoblotting (WB). Proteins were revealed using antibodies against c-Myc, β-actin, or HIV-1 PR
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4546280&req=5

Fig4: Identification of the PR cleavage site in RIPK1. a Primary sequence of human RIPK1 (aa 449–476 according to NCBI Reference Sequence NP_003795.2) with PR cleavage site between residues 462 and 463. Indicated point mutations were introduced by Quikchange mutatgenesis. b HEK293T cells were transfected with expression plasmids encoding wild-type (wt) RIPK1, RIPK1 with a double-mutation in the PR cleavage site (RIPK1 PRres), or RIPK1 with a non-sense mutation in the PR cleavage site (RIPK1 ΔC), respectively, along with catalytically active HIV PR (5 ng/well) in the absence (−) or presence (+) of SQV (5 μM). Cell lysates were subjected to SDS-PAGE and immunoblotting (WB). Proteins were revealed using antibodies against c-Myc, β-actin, or HIV-1 PR

Mentions: We next used mass spectrometry to identify the precise cleavage site in RIPK1. To do so, we co-expressed Myc-tagged RIPK1 and PR in HEK293T cells. The N-terminal fragment of cleaved RIPK1 was purified from total cell extracts by immunoprecipitation with an anti-Myc antibody and purified complexes were subjected to SDS-PAGE analysis. A prominent band corresponding to the N-terminal cleavage product was submitted to mass spectrometry (Additional file 9: Figure S7A). Analysis revealed the presence of a non-tryptic peptide, corresponding to amino acids 436–462 of human RIPK1. This indicates that the cleavage site is located in the ID region of RIPK1 (Fig. 4a).Fig. 4


HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2.

Wagner RN, Reed JC, Chanda SK - Retrovirology (2015)

Identification of the PR cleavage site in RIPK1. a Primary sequence of human RIPK1 (aa 449–476 according to NCBI Reference Sequence NP_003795.2) with PR cleavage site between residues 462 and 463. Indicated point mutations were introduced by Quikchange mutatgenesis. b HEK293T cells were transfected with expression plasmids encoding wild-type (wt) RIPK1, RIPK1 with a double-mutation in the PR cleavage site (RIPK1 PRres), or RIPK1 with a non-sense mutation in the PR cleavage site (RIPK1 ΔC), respectively, along with catalytically active HIV PR (5 ng/well) in the absence (−) or presence (+) of SQV (5 μM). Cell lysates were subjected to SDS-PAGE and immunoblotting (WB). Proteins were revealed using antibodies against c-Myc, β-actin, or HIV-1 PR
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4546280&req=5

Fig4: Identification of the PR cleavage site in RIPK1. a Primary sequence of human RIPK1 (aa 449–476 according to NCBI Reference Sequence NP_003795.2) with PR cleavage site between residues 462 and 463. Indicated point mutations were introduced by Quikchange mutatgenesis. b HEK293T cells were transfected with expression plasmids encoding wild-type (wt) RIPK1, RIPK1 with a double-mutation in the PR cleavage site (RIPK1 PRres), or RIPK1 with a non-sense mutation in the PR cleavage site (RIPK1 ΔC), respectively, along with catalytically active HIV PR (5 ng/well) in the absence (−) or presence (+) of SQV (5 μM). Cell lysates were subjected to SDS-PAGE and immunoblotting (WB). Proteins were revealed using antibodies against c-Myc, β-actin, or HIV-1 PR
Mentions: We next used mass spectrometry to identify the precise cleavage site in RIPK1. To do so, we co-expressed Myc-tagged RIPK1 and PR in HEK293T cells. The N-terminal fragment of cleaved RIPK1 was purified from total cell extracts by immunoprecipitation with an anti-Myc antibody and purified complexes were subjected to SDS-PAGE analysis. A prominent band corresponding to the N-terminal cleavage product was submitted to mass spectrometry (Additional file 9: Figure S7A). Analysis revealed the presence of a non-tryptic peptide, corresponding to amino acids 436–462 of human RIPK1. This indicates that the cleavage site is located in the ID region of RIPK1 (Fig. 4a).Fig. 4

Bottom Line: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR.Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB.These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA. rwagner@sbpdiscovery.org.

ABSTRACT

Background: HIV-1 protease (PR) is essential for viral infectivity as it cleaves Gag and Gag-Pol polyprotein precursors during viral maturation. Recent evidence suggests that cellular proteins can also be cleaved by PR, perhaps representing an important viral strategy to counter host defense mechanisms. Receptor-interacting protein kinase 1 (RIPK1) and RIPK2 belong to a family of serine/threonine kinases with conserved domain architecture and important functions in apoptosis, necrosis and innate immunity.

Results: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR. In RIPK1, we identified a putative PR cleavage site; a mutation at this site rendered RIPK1 resistant to PR cleavage. RIPK1 and RIPK2 were cleaved during HIV-1 infection of T cell lines or primary activated CD4(+) T cells. Interfering with the viral life cycle at different stages by the addition of specific inhibitors against RT, integrase, or PR, completely prevented RIPK1 and RIPK2 cleavage. Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB.

Conclusions: These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.

No MeSH data available.


Related in: MedlinePlus