Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites Of

Article Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration Graphical Abstract Authors Vasudevan Achuthan, Jill M. Perreira, Gregory A. Sowd, ..., Stefan G. Sarafianos, Abraham L. Brass, Alan N. Engelman Correspondence [email protected] (A.L.B.), alan_engelman@dfci. harvard.edu (A.N.E.) In Brief Prior work indicated that the nuclear periphery dictated HIV-1 integration site selection. Using multiple orthologous approaches, Achuthan et al. fail to garner evidence for preferential targeting of the periphery. The interaction between viral capsid and CPSF6 enables HIV-1 to bypass integration into peripheral heterochromatin and penetrate the nuclear structure for integration. Highlights d CA-CPSF6 interaction as opposed to nuclear periphery dictates HIV-1 integration d CPSF6 enables HIV-1 to penetrate the nuclear interior beyond the nuclear periphery d Loss of CPSF6 interaction results in integration at lamina- associated domains d LEDGF/p75 does not play a significant role in intranuclear HIV-1 localization Achuthan et al., 2018, Cell Host & Microbe 24, 392–404 September 12, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.chom.2018.08.002 Cell Host & Microbe Article Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration Vasudevan Achuthan,1,2 Jill M. Perreira,3 Gregory A. Sowd,1,2 Maritza Puray-Chavez,4 William M. McDougall,3 Adriana Paulucci-Holthauzen,5 Xiaolin Wu,6 Hind J. Fadel,7 Eric M. Poeschla,8 Asha S. Multani,5 Stephen H. Hughes,9 Stefan G. Sarafianos,4,11 Abraham L. Brass,3,10,* and Alan N. Engelman1,2,12,* 1Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA 2Department of Medicine, Harvard Medical School, Boston, MA 02115, USA 3Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA 4Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USA 5Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 6Leidos Biomedical Research, Inc., Frederick, MD 21702, USA 7Division of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA 8Division of Infectious Diseases, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA 9HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA 10Gastroenterology Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA 11Present address: Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30332, USA 12Lead Contact *Correspondence: [email protected] (A.L.B.), [email protected] (A.N.E.) https://doi.org/10.1016/j.chom.2018.08.002 SUMMARY tivity, promoters/enhancers, histone modifications, and gene-rich regions of chromatin (reviewed in Craigie and Bushman, 2014; HIV-1 integration into the host genome favors Serrao and Engelman, 2016). HIV-1 integration favors actively actively transcribed genes. Prior work indicated transcribed genes that reside within relatively gene-dense regions that the nuclear periphery provides the architectural (Schroder et al., 2002), a phenotype that depends in large part on basis for integration site selection, with viral the interactions of two viral proteins, integrase (IN) and capsid capsid-binding host cofactor CPSF6 and viral inte- (CA), with respective cellular binding partners lens epithelium- grase-binding cofactor LEDGF/p75 contributing to derived growth factor (LEDGF)/p75 (Cherepanov et al., 2003; Ciuffi et al., 2005; Marshall et al., 2007; Shun et al., 2007b; Singh et al., selection of individual sites. Here, by investigating 2015) and cleavage and polyadenylation specificity factor 6 the early phase of infection, we determine that (CPSF6) (Lee et al., 2010; Sowd et al., 2016). LEDGF/p75 and HIV-1 traffics throughout the nucleus for integration. CPSF6, however, influence HIV-1 integration in different ways. CPSF6-capsid interactions allow the virus to bypass LEDGF/p75 depletion preferentially reduces integration into peripheral heterochromatin and penetrate the nu- genes as compared with gene-dense regions (Ciuffi et al., 2005; clear structure for integration. Loss of interaction Koh et al., 2013; Marshall et al., 2007; Ocwieja et al., 2011; Shun with CPSF6 dramatically alters virus localization et al., 2007b) and shifts intragenic sites toward 50 end regions toward the nuclear periphery and integration into (Shun et al., 2007b; Singh et al., 2015;Sowd et al., 2016), indicating transcriptionally repressed lamina-associated het- that LEDGF/p75 primarily functions to position integration along erochromatin, while loss of LEDGF/p75 does not gene bodies. Although CPSF6 knockout reduces intragenic inte- significantly affect intranuclear HIV-1 localization. gration more dramatically than LEDGF/p75 depletion, positional targeting within transcription units is relatively random. By Thus, CPSF6 serves as a master regulator of HIV-1 contrast, HIV-1 dramatically loses preference for integration near intranuclear localization by trafficking viral preinte- activating epigenetic marks and favors gene-sparse regions, indi- gration complexes away from heterochromatin at cating that CPSF6 predominantly shields HIV-1 from heterochro- the periphery toward gene-dense chromosomal re- matin (Sowd et al., 2016). CPSF6 facilitates HIV-1 PIC nuclear gions within the nuclear interior. import (Chin et al., 2015; Dharan et al., 2016; Peng et al., 2014), and other import cofactors, including transportin 3 (TNPO3), nucleoporin 358 (NUP358) (Ocwieja et al., 2011), cyclophilin A INTRODUCTION (Schaller et al., 2011), and NUP153 (Di Nunzio et al., 2013; Koh et al., 2013), can also influence sites of integration, indicating a po- HIV-1 replication initiates with viral attachment and cell entry, fol- tential link between PIC nuclear import and integration targeting lowed by reverse transcription, nuclear import of the preintegra- (Di Nunzio, 2013; Ko¨ nig et al., 2008). tion complex (PIC), and viral DNA (vDNA) integration. Integration Image-based studies have been used to map positions of into cell genomes is non-random, with different types of retrovi- PICs, integrated proviruses, and favored gene targets (recurrent ruses displaying distinct preferences for genes, transcriptional ac- integration genes [RIGs]) within cell nuclei. Such analyses are 392 Cell Host & Microbe 24, 392–404, September 12, 2018 ª 2018 Elsevier Inc. facilitated by determining relative distance of the imaged focus 2015) in intranuclear HIV-1 localization depleted the proteins by from the nuclear edge, and binning results into three concentric RNAi, although Burdick et al. (2017) analyzed HeLa cells with zones of equal area: peripheral nuclear (PN), mid-nuclear (MN), genomic deletions of the LEDGF/p75 coding gene PSIP1.To and central nuclear (CN) (Chin et al., 2015; Marini et al., 2015). systematically address the roles of LEDGF/p75 versus CPSF6, For studies that did not perform such analyses, we have for the we initially used WT HEK293T cells and isogenic derivatives sake of comparison correlated reported distance measures to knocked out for PSIP1 (LEDGF/p75 knockout [LKO]) (Fadel zonal region (STAR Methods). HIV-1 PICs (Albanese et al., et al., 2014), CPSF6 (CKO), or both PSIP1 and CPSF6 (double 2008; Burdick et al., 2013, 2017; Francis and Melikyan, 2018) KO [DKO]) (Sowd et al., 2016). Cells infected with VSV-G-pseu- and proviruses (Di Primio et al., 2013; Marini et al., 2015) predom- dotyped HIV-1NL4-3 at the approximate MOI of 350 were imaged inantly mapped to the PN and MN. RIGs were mapped by fluores- at 24 hr post-infection (hpi) using the Provirus ViewHIV assay that cence in situ hybridization (FISH) also to PN and MN areas, with predominantly detects integrated virus (Chin et al., 2015)(Fig- some PN loci in proximity to nuclear pore complexes (NPCs) ure S1). DNA signals from confocal microscopy z stacks were (Marini et al., 2015). Such observations have led to the suggestion binned into PN, MN, and CN areas. In both WT and LKO cells, that active chromatin at the nuclear periphery determines the vDNA dispersed fairly equally across the nuclear sections (Fig- architectural basis for HIV-1 integration site selection (Marini ure 1A). In line with our knockdown studies (Chin et al., 2015), et al., 2015). However, the roles of known integration targeting HIV-1 DNA significantly relocalized to the PN in CKO cells cofactors in this process, such as LEDGF/p75 and CPSF6, (76.5% ± 1.4%) with concomitant reductions in MN and CN require clarification. For example, prior studies disagree as to a localization. Consistent with a dominant role for CPSF6 in deter- potential role for LEDGF/p75: whereas two reports indicated mining intranuclear HIV-1 localization, 65.1% ± 0.9% of vDNA that LEDGF/p75 played a role in PN targeting (Marini et al., signals mapped to the PN area of DKO cell nuclei (Figure 1A). 2015; Vranckx et al., 2016), two other papers concluded To assess the generalizability of these observations, we uti- LEDGF/p75 does not contribute to the macrolocalization of lized the MICDDRP bDNA hybridization technique, which, at HIV-1 within the nucleus (Burdick et al., 2017; Quercioli et al., 24 hpi, does not distinguish between unintegrated vDNA and 2016). CPSF6, by contrast, was reportedly important for nuclear proviral DNA (Puray-Chavez et al., 2017). Cells infected at MOI penetration (Chin

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