Research article

Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

Jörg Votteler¹ , Elena Iavnilovitch² , Orit Fingrut² , Vivian Shemesh² , Daniel Taglicht² , Omri Erez² , Stefan Sörgel¹ , Torsten Walther³ , Norbert Bannert⁴ , Ulrich Schubert^1,3 and Yuval Reiss²

¹Institute of Virology, Friedrich-Alexander University, Erlangen, Germany

²Proteologics Ltd, Rehovot, Israel

³ViroLogik GmbH, Erlangen, Germany

⁴Robert Koch Institute, Berlin, Germany

author email corresponding author email

BMC Biochemistry 2009, 10:12doi:10.1186/1471-2091-10-12

Published:	24 April 2009

Abstract

Background

The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila.

Results

In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1Δ_PTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSH^V14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1Δ_PTAP L-domain mutant (YPX_nL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPX_nL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.

Conclusion

Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPX_nL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.