Identification of cellular factors binding to acetylated HIV-1 integrase.
The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle.
Allouch
Awatef
A
Molecular Biology Laboratory, Scuola Normale Superiore, Pisa, Italy. a.allouch@sns.it
Cereseto
Anna
A
D000107:Acetylation; D002460:Cell Line; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D058605:Host-Derived Cellular Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D058605:Host-Derived Cellular Factors; D011506:Proteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1