Abstract
The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in virus replication and infectivity. Here we show that Vif is phosphorylated and regulated by p44/42 mitogen-activated protein kinase (MAPK). Vif phosphorylation by MAPK was demonstrated in vitro as well as in vivo and was shown to occur on serine and threonine residues. Two-dimensional tryptic phosphopeptide mapping indicated that Vif is phosphorylated by MAPK on the same sites in vitro and in vivo. Radioactive peptide sequencing identified two phosphorylation sites, Thr96 and Ser165. These phosphorylation sites do not correspond to the known optimum consensus sequences for phosphorylation by MAPK (PX(S/T)P) nor to the minimum consensus sequence ((S/T)P), indicating that MAPK can phosphorylate proteins at sites other than those containing the PX(S/T)P or (S/T)P motifs. Synthetic Vif peptides corresponding to the local sequences of the phosphorylation sites were not phosphorylated by MAPK, suggesting that recognition of these sites by MAPK is likely to require structural determinants outside the phosphorylation site. Mutations of the Thr96 site, which is conserved among Vif sequences from HIV-1, HIV-2, and SIV, resulted in significant loss of Vif activity and inhibition of HIV-1 replication. These results suggest that MAPK plays a direct role in regulating HIV-1 replication and infectivity by phosphorylating Vif and identify a novel mechanism for activation of HIV-1 replication by mitogens and other extracellular stimuli.
Highlights
The Vif protein of human immunodeficiency virus type 1 (HIV-1)1 and other lentiviruses is essential for virus replication in peripheral blood T lymphocytes in vitro and in vivo [1,2,3,4,5]
Previous studies have shown that the HIV-1 p17 Gag, p24 Gag, Vif, Vpu, Rev, and Nef proteins are phosphorylated by cellular kinases in vitro and in vivo [15, 17,18,19,20,21,22,23,24,25,26]. p17 Gag, Nef, and Rev are phosphorylated on serine/threonine residues by protein kinase C [17, 19, 22], and Vpu is phosphorylated on serine by casein kinase II [23]
We show that HIV-1 Vif is phosphorylated on Thr96 and Ser165 by p44/42 mitogen-activated protein kinase both in vitro and in vivo
Summary
Materials—Rabbit anti-ERK1 and anti-ERK2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An aliquot of immunoprecipitated MAPK (12.5 l), recombinant p42 MAPK (50 ng), or CEM cell lysate (2 g) was incubated for 30 min at room temperature with kinase reaction mixture (2 g of MBP or 2 g of recombinant Vif protein, 20 M ATP, and 2.5–5.0 Ci of [␥-32P]ATP in kinase buffer). Approximately 20 g of CEM cell lysate was subjected to electrophoresis on a 10% SDS-polyacrylamide gel containing 500 g of recombinant Vif/ml or 200 g of MBP/ml co-polymerized in the separating gel. Samples with equivalent amounts of protein (5–10 g) were resolved by 15% SDSPAGE and transferred to PVDF membrane for immunoblotting with rabbit anti-ERK1 or anti-ERK1 and anti-ERK2 (1:1500 dilution of each), rabbit anti-phosphorylated MAPK (1:1000 dilution), or rabbit anti-Vif (1:2000 dilution) [15] using the ECL system (Amersham Pharmacia Biotech) as described [15]. Data were analyzed by analysis of variance with post hoc Tukey-Kramer test and are expressed as means Ϯ S.D
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