Abstract
The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6(gag) protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015-1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6(gag) phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6(gag). Substitution by an alanine of the Thr(23) phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55(gag) maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6(gag) protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle.
Highlights
Soon after the viral particle pinches off and is released in the extracellular compartment, the viral protease becomes activated and the Pr55gag polyprotein is cleaved into MAp17gag, CAp24gag, NCp7gag, and p6gag proteins and p1, p2 spacer peptides
In addition to MAp17gag and CAp24gag that have previously been reported to be phosphorylated by protein kinases packaged within viral particles [32, 33], we report here that the p6gag protein and its p15gag and Pr55gag polyprotein precursors are phosphorylated by protein kinase(s) incorporated within human immunodeficiency virus type 1 (HIV-1) virions
Using GSTfused proteins, p6gag was found to be phosphorylated by ERK-2, a host cell MAP kinase (MAPK) that we previously found to be incorporated within HIV-1 particles [36]
Summary
5Ј-GCTCTGGTCTGGCCTGGGATCCACGC 5Ј-CTGGTGGGGCTGCTGGCTCTGGTCTG 5Ј-CCAAACCTGAAGGCCTCTTCTGGTG 5Ј-GAGGGAGTTGTTGCCTCTTCCCCAAAG 5Ј-CTGAGAGGGAGTTGCTGTCTCTTCC 5Ј-CTGAGAGGGAGCTGTTGTCTCTTCC 5Ј-CTCCTGCTTCTGAGCGGGAGTTGTTG 5Ј-AAGAGTGATCTGAGGGCAGCTAAAGG 5Ј-GCTGCCAAAGAGTGCTCTGAGGGAAG 5Ј-GACGAGGGGTCGGCGCCAAAGAGTG 5Ј-TTATTGTGACGCGGGGTCGCTGCCAAAG 5Ј-TTATTGTGCCGAGGGGTCGCTGCCAAAG. 5Ј-CTTCTGAGAGGGAGCTGTTGTCTCTT. For CAp24gag, three distinct phosphorylation sites on serine residues were identified [33] As mutations at these sites were observed to block HIV-1 replicative cycle at an early reverse transcription step, CAp24gag phosphorylation was proposed to contribute to the early post-entry step by promoting the dissociation of the viral core. A faint number of particles released from the producing cells was isolated that were found to display altered morphology and size Such particles were characterized by incomplete maturation of Pr55gag precursors accompanied by modifications in the incorporation of reverse transcriptase subunits. Our data indicate that phosphorylation of a unique site of the p6gag domain by ERK-2 plays a critical role in the late stage of the HIV-1 life cycle, by contributing to the regulation of viral assembly and/or release
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