The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes

Sheeba Rasheedi,Ming-Chieh Shun,Erik Serrao,Gregory A Sowd,Juan Qian,Caili Hao,Twishasri Dasgupta,Alan N Engelman,Jacek Skowronski

doi:10.1074/jbc.m116.721647

Abstract

HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

Highlights

Integration of the DNA copy of the viral RNA into chromosomal DNA of the host cell is an integral step within the retrovirus replication cycle
cleavage and polyadenylation specificity factor 6 (CPSF6) Is Sequestered into the cleavage factor Im (CFIm) Complex—CPSF6 is a subunit of a heterotetrameric CFIm complex (Fig. 1A)
Separation of the eluates by SDS-PAGE followed by immunoblotting revealed that CPSF6 co-precipitated the composed of two 25-kDa (CPSF5) and CPSF7 CFIm subunits from extracts of both U937 and Jurkat T cells as expected (Fig. 1B)

Summary

Introduction

Integration of the DNA copy of the viral RNA into chromosomal DNA of the host cell is an integral step within the retrovirus replication cycle. HIV-1 preferentially integrates into active genes located in gene dense chromosomal regions [5] This pathway was initially linked to the binding of the cleavage and polyadenylation specificity factor 6 (CPSF6) protein, a pre-mRNA splicing factor and member of the serine/arginine-rich (SR) protein family, to the viral capsid before the PIC translocation to the host cell nucleus (6 –10). Silencing expression of any of the above proteins abolishes the characteristic pattern of preferential HIV-1 integration into active genes [3, 12, 13, 18] These latter findings implicate these proteins as elements of a linear pathway that commits the HIV-1 reverse transcription complex/PIC to a particular nuclear import venue that controls PIC delivery to active chromatin. CPSF6 and CPSF7 display a very similar overall domain organization, they probably play non-redundant roles in CFIm complex and mRNA 3Ј end processing, as they have distinct interaction partners linking them to transcription and RNA processing machineries (34 –37)

Methods

Results

Conclusion