https://orcid.org/0000-0001-5665-9195
Figures
Abstract
The early stages of HIV-1 infection include the trafficking of the viral core into the nucleus of infected cells. However, much remains to be understood about how HIV-1 accomplishes nuclear import and the consequences of the import pathways utilized on nuclear events. The host factor cleavage and polyadenylation specificity factor 6 (CPSF6) assists HIV-1 nuclear localization and post-entry integration targeting. Here, we used a CPSF6 truncation mutant lacking a functional nuclear localization signal (NLS), CPSF6-358, and appended heterologous NLSs to rescue nuclear localization. We show that some, but not all, NLSs drive CPSF6-358 into the nucleus. Interestingly, we found that some nuclear localized CPSF6-NLS chimeras supported inefficient HIV-1 infection. We found that HIV-1 still enters the nucleus in these cell lines but fails to traffic to speckle-associated domains (SPADs). Additionally, we show that HIV-1 fails to efficiently integrate in these cell lines. Collectively, our results demonstrate that the NLS of CPSF6 facilitates steps of HIV-1 infection subsequent to nuclear import and additionally identify the ability of canonical NLS sequences to influence cargo localization in the nucleus following nuclear import.
Author summary
During HIV-1 infection, the viral capsid, which encloses the viral genome and essential proteins required for reverse transcription and integration, traffics towards the nucleus and enters through the nuclear pore complex (NPC). Following entry into the nucleus, reverse transcription is completed and viral capsid disassembles for integration of the viral DNA into a host chromosome. In this study, we investigated the role of the capsid-binding host factor CPSF6 in these events, specifically focusing on its nuclear localization signal (NLS) located at the protein’s C-terminus. Swapping the NLS for heterologous NLSs from other proteins significantly impacted HIV-1’s ability to infect cells. For NLSs that conveyed CPSF6 nuclear import, we further showed this defect in infection occurred at the level of viral DNA integration. This study highlights the importance of the NLS in CPSF6 to dictate nuclear import pathways that are congruent with functional HIV-1 infection. By extension, our study emphasizes that different NLSs can have comparatively dramatic post-nuclear effects on host cargo function.
Citation: Rohlfes N, Radhakrishnan R, Singh PK, Bedwell GJ, Engelman AN, Dharan A, et al. (2025) The nuclear localization signal of CPSF6 governs post-nuclear import steps of HIV-1 infection. PLoS Pathog 21(1): e1012354. https://doi.org/10.1371/journal.ppat.1012354
Editor: Li Wu, University of Iowa, UNITED STATES OF AMERICA
Received: June 18, 2024; Accepted: January 5, 2025; Published: January 17, 2025
Copyright: © 2025 Rohlfes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Illumina Sequencing results can be found with Accession no: PRJNA1123763. All other relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by the National Institute of Health (NIH, https://www.nih.gov) grant R01AI162694 to EMC and R01AI052014 and U54AI170791 awarded to ANE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
A defining feature of lentiviruses, including human immunodeficiency virus-1 (HIV-1), is the ability to infect non-dividing cells, which requires import of the viral ribonucleoprotein complex (vRNPC) into the target cell nucleus. This requires that the virus traverse nuclear pore complexes (NPCs) that exist within the nuclear membranes of non-dividing cells. NPCs are large, macromolecular complexes composed of multiple copies of about 30 nucleoporins (Nups) [1–3]. Critically, NPCs are responsible for maintaining the nuclear permeability barrier, which restricts entry of many proteins while simultaneously allowing directed trafficking of hundreds to thousands of cargoes per second [4,5]. As a result, nucleocytoplasmic trafficking is a complex and tightly regulated process. To ensure the correct cargo is being transported into the nucleus, short amino acid sequences called nuclear localization signals (NLSs) are recognized by nuclear transport receptors to facilitate nuclear import [6,7].
The HIV-1 capsid core is comprised of approximately 250 hexamers and exactly 12 pentamers of capsid protein (CA) forming a conical structure that houses the viral genome and essential enzymes [8–12]. CA is the primary viral determinant driving nuclear import [13], and CA accordingly interacts with several Nups including Nup358 [14–16], Nup153 [15,17–22], Nup58 [15,21], Nup98 [15,21], Nup88 [23], and Nup62 [15,20]. Capsid additionally interacts with cleavage and polyadenylation specificity factor 6 (CPSF6) [15,17,19,21,24–26], which is a component of the cleavage factor I mammalian (CFIm) complex that helps to dictate pre-mRNA polyadenylation site selection [27]. Following trafficking through the cytoplasm, the core docks at the cytoplasmic face of the NPC via interactions with Nup358 [16,28]. This promotes passage through the NPC [16,28,29], leading to docking interactions with Nup153 on the nucleoplasmic side of the NPC [20], where interactions with CPSF6 are thought to release the core into the nucleus [30,31]. CPSF6 traffics with the core further into the nucleus to subnuclear bodies called nuclear speckles (NSs) [30,32–34]. NSs are found near gene-dense regions of chromatin and prior work has shown that HIV-1 preferentially targets these regions for viral DNA integration [33,35]. Importantly, HIV-1 capsid mutants defective for CPSF6 binding display aberrant integration site selection, highlighting the impact of CPSF6 for integration [16,36].
CPSF6 contains a functional NLS at its C-terminus, and as a result is predominantly found in the nucleus of cells [26,37,38]. CPSF6 is expressed as two splice isoforms. Isoform 1, the major form, is composed of 551 residues while isoform 2, containing 588 amino acids, carries internal residues encoded by exon 6 that are removed from isoform 1 by alternative splicing [26]. A truncation of CPSF6 isoform 2 containing amino acids 1–358 (CPSF6-358) was identified by the KewalRamani group, where they noted the protein was mislocalized to the cytoplasm and acted as an artificial restriction factor [26]. This finding led to identification of the N74D capsid mutant, which is defective in its ability to bind to CPSF6 [26]. Moreover, N74D CA mutant and wildtype (WT) CA viruses showed variable sensitivities to Nup knockdowns, highlighting that the mutant virus utilized a different nuclear import pathway than the WT [26]. Investigation of the P90A capsid mutant, which disrupts binding of cyclophilin A (CypA) to CA, also found that it utilizes a nuclear import pathway distinct from WT virus [16,39]. Our previous studies using a nuclear pore blockade have also supported the idea of multiple nuclear import pathways [40]. We observed that infection mediated by WT HIV-1 was potently inhibited by Nup62 blockade, but infection mediated by N74D and P90A CA mutant viruses was comparatively less sensitive or insensitive to the Nup62-mediated NPC blockade, suggesting the utilization of distinct nuclear import pathways, likely utilizing different NPCs [40]. Recently, several groups reported that NLS swapping can alter sensitivity to the restriction factor MX2 [23,41]. Kane et al. reported that appending some NLSs to a GFP-LacZ fusion resulted in MX2-mediated restriction of nuclear import, while other NLSs resulted in GFP-LacZ nuclear import that was insensitive to MX2 restriction [23]. Chai et al. modified MX2’s NLS by appending several heterologous NLSs to the N-terminus of MX2. They found that only some of these NLS-MX2 chimeras retained MX2’s ability to restrict nuclear import of HIV-1 [41]. These findings showed that some, but not all, import pathways retained MX2 restriction activity and that the NLS determined which pathway was used.
To better understand the role of CPSF6’s NLS, we attached heterologous NLSs to CPSF6-358 to determine the impact of divergent NLSs on nuclear import and infection by HIV-1 and related lentiviruses. We observed that the addition of NLS sequences that restored the nuclear localization of CPSF6 also promoted the nuclear import of HIV-1 during infection. However, despite effective nuclear import, we noted that some NLSs promoted a dysfunctional form of nuclear import that did not facilitate or altered subsequent intranuclear steps of infection, including integration. These results demonstrate that the NLS promoting the nuclear import of HIV-1 can impact the nuclear stages of infection. Previous reports have shown that CPSF6’s C-terminal intrinsically disordered region (IDR), which is the protein’s NLS [38], confers NS colocalization and condensation to heterologous fusion partners such as green fluorescent protein (GFP) [37,42,43]. Our results support these findings, as we found only the full-length CPSF6, which contains an intact IDR, formed condensates following HIV-1 infection. However, our findings show re-localization to NSs can occur independently of the IDR, as CPSF6-NLS constructs containing the SV40 or C-MYC NLS effectively trafficked the HIV-1 RNPC to NSs despite lacking the IDR. These data show that specific NLSs license subsequent trafficking and integration by lentiviruses and further refines our understanding of the nuclear import pathways utilized by HIV-1. These data also provide evidence that NLSs possess the ability to influence post-nuclear import activities of their cargoes.
Results
Heterologous NLSs can confer CPSF6-358 nuclear localization
To understand more about CPSF6’s NLS and how it influences post-nuclear HIV-1 biology, we used the well described truncation mutant containing amino acids 1–358 (CPSF6-358), which lacks the C-terminal IDR, also called an RS-like domain (RSLD) [26]. Because CPSF6-358 was initially derived from exon 6-containing isoform 2 [26], our isoform 1-based version harbors 321 total residues. The RSLD contains the functional NLS for CPSF6, and as a result, CPSF6-358 displays a significant cytoplasmic localization, in contrast to full-length CPSF6, which is predominantly nuclear [26,38]. We then introduced various NLS’s at the C-terminus of CPSF6-358 to rescue nuclear localization through several distinct nuclear import pathways (Fig 1A and Table 1). We chose representative NLSs, which were either cellular or viral in origin, and that spanned across the different NLS classifications. To understand the impact of these NLSs on infection, we first knocked out endogenous CPSF6 in HeLa cells and generated stable cell lines expressing the indicated CPSF6-NLS chimeras (Fig 1B and 1C). We observed that several of the NLSs (SV40, C-MYC, NP, Rac3, HNRNP K, HNRNP A1, and MX2) effectively rescued nuclear localization of our CPSF6-358 construct (Fig 1D and Table 1). Notably, some NLSs (DDX21, RB, HTLV-1 Rex, and UL79) were unable to confer effective nuclear localization, and the corresponding CPSF6-358 chimeric proteins retained cytoplasmic distribution (Fig 1D and 1E and Table 1). These results are in line with previous studies of MX2 localization, which showed variable degrees of nuclear localization of heterologous MX2 constructs across the tested NLSs [23,41]. Overall, some, but not all, of our chimeric CPSF6-NLS constructs functionally localized to HeLa cell nuclei.
(A) Schematic design of chimeric CPSF6-NLS constructs showing NLSs being fused to the C-terminus of CPSF6-358 derived from human isoform 1. (B) Western blot analysis of HeLa cells using anti-CPSF6 antibody to confirm CRISPR/Cas9-mediated knockout of endogenous CPSF6. (C) Western blot analysis of stably transduced HeLa cells using anti-CPSF6 antibody to detect CPSF6-NLS construct expression following 48 h doxycycline induction. Anti-GAPDH antibody used as loading control. (D) Immunofluorescent microscopic images stained using anti-HA antibodies (green) and Hoechst dye (blue) to visualize CPSF6-NLS construct localization. Representative of 3 independent experiments with at least 10 images per experiment. (E) Percent nuclear signal was determined using ImageJ. Statistical analysis in E was determined using one-way ANOVA. Significant differences are indicated: **** P<0.0001. RRM–RNA Recognition Motif. PRD–Proline Rich Domain. RSLD–Arginine Serine-Like Domain. HA–Hemagglutinin tag. NLS–Nuclear Localization Signal. CPSF6-FL–Full Length CPSF6.
Of note, our CPSF6-358, as well as the CPSF6-NLS chimeras with significant cytoplasmic distributions, exhibited partial nuclear accumulation, which was previously described for original isoform 2 CPSF6-358, as well as a similar isoform 1 truncation known as CPSF6-375 [26,44]. These C-terminal deletion mutants retain the N-terminal RRM domain that mediates binding of CPSF6 to its CFIm partner protein CPSF5 (Fig 1A) [37]. CFIm in turn functions to regulate polyadenylation in the context of a much larger cleavage and polyadenylation (CPA) complex [45]. We speculate that partial nuclear accumulation of CPSF6-358 and non-functional NLS derivatives is due to CFIm-mediated associated with CPA as a consequence of cell line construction, which necessitated multiple rounds of cell division and nuclear reorganization.
Nuclear localization of CPSF6-358 does not uniformly rescue HIV-1 infection
CPSF6 is an HIV-1 host factor that binds directly to the HIV-1 capsid to aid nuclear entry of the viral capsid and integration site targeting [17,24–26,30,31,35,54]. To further understand if changing CPSF6’s NLS impacts HIV-1 infection, we utilized single-round HIV-1 luciferase reporter virus to infect our chimeric CPSF6 cell lines, and infectivity was determined as a measurement of viral promoter activity driving reporter gene expression. To highlight phenotypes associated with NLS functionality, the predominance of experiments reported here were conducted with growth-arrested cells. Consistent with previous studies that depleted cellular CPSF6 via RNA interference [26], CPSF6-KO did not perturb HIV-1 infectivity in HeLa cells, and expression of CPSF6-358 in CPSF6-KO cells potently inhibited infection (Fig 2A). As might be expected, a similar inhibition was observed in cells expressing chimeric NLSs that failed to confer CPSF6-358 nuclear localization (DDX21, RB, HTLV-1 Rex, and UL79) (Fig 2A, striped bars). We, moreover, observed three distinct phenotypes amongst cells expressing CPSF6 chimeras with restored nuclear localization. We found that the C-MYC and Rac3 NLS not only fully rescued infection, but resulted in a small but significant increase in infectivity compared to the infection observed in CPSF6-KO and CPSF6-FL cells (Fig 2A). In contrast, the SV40, HNRNP K, and HNRNP A1 NLSs rescued infection to levels that were indistinguishable from CPSF6-FL (Fig 2A). Finally, HIV-1 infection was significantly reduced in cells expressing the CPSF6 chimeras with NP and MX2 NLSs (Fig 2A), despite these NLSs conferring effective CPSF6-358 nuclear localization. These phenotypes persisted across several viral titrations, showing that the differences in infectivity were concentration independent (Fig 2B). We observed similar infectivity findings using a fluorescent reporter virus, revealing that the results were independent of the viral reporter gene utilized (Fig 2C). Additionally, these results were independent of cell cycle arrest, as we observed similar phenotypes when cells were not growth arrested with aphidicolin (Fig 2D). These results show that simply conferring constitutive CPSF6-358 nuclear localization is insufficient to restore HIV-1 infectivity and is impacted by the NLS used to mediate CPSF6-358 nuclear localization.
(A) WT HIV-1 firefly luciferase reporter virus infectivity was measured 48 h post-infection in growth arrested HeLa cells. Infectivity was normalized to cells expressing full-length CPSF6. Striped bars represent constructs with cytoplasmic CPSF6-358-NLS fractions. (B) Serial dilutions show that infectivity phenotypes hold across several viral concentrations in growth arrested cells. (C) WT HIV-1 mCherry reporter virus was used to infect growth arrested HeLa cell lines and infectivity was determined using flow cytometry. (D) Dividing HeLa cells infected with WT HIV-1 firefly luciferase reporter virus display comparable phenotypes to non-dividing cells. Results (mean ± SEM) are representative from at least 3 independent experiments with at least technical duplicate samples per experiment. Statistical analysis was determined using one-way ANOVA. Significant differences are indicated: * P<0.05, ** P<0.01, **** P<0.0001. CPSF6-FL–Full Length CPSF6.
NLSs are categorized into classes based on the structure of the NLS sequence [55]. We accordingly wondered if NLS classification could explain the phenotypic differences observed amongst the CPSF6-NLS chimeras. However, the ability to influence infection positively or negatively was not dependent on the type of NLS. RB, Rac3, and NP are three examples of bipartite NLSs that conferred three distinct phenotypes under our assay conditions (Table 1). Rac3 and NP conferred CPSF6-358 nuclear localization, whereas the RB NLS chimera remained partially cytoplasmic. Further, Rac3 fully rescued infectivity, while NP resulted in an infectivity defect. Additionally, we tested two PY-NLSs, HNRNP A1 and UL79, and found that HNRNP A1 conferred CPSF6-358 nuclear localization and supported HIV-1 infection similarly as CPSF6-FL, while UL79 failed to rescue both CPSF6-358 nuclear localization and HIV-1 infection (Table 1). These findings suggest that the ability to influence HIV-1 infection cannot be explained by NLS classification, and that NLSs belonging to the same classification, such as bipartite or PY-NLS, functioned differently from others within the same NLS class. Together, these results display the importance of the nuclear import pathway utilized by CPSF6 for productive HIV-1 infection.
CPSF6-NLS chimeras show virus-specific influences on infection
To ensure that the observed changes in viral gene expression were driven through CA-CPSF6 interactions and not a deleterious effect of chimeric protein expression on cell function, we infected our cells with the CPSF6 binding deficient HIV-1 CA mutant N74D [26]. The Asn>Asp substitution in CA occurs within the CPSF6 binding pocket, rendering CA unable to bind to CPSF6. Importantly, this CA mutant retains the ability to productively infect HeLa cells [26]. When we infected our chimeric cell lines with N74D virus, we observed no differences in viral gene expression, demonstrating that the differences in WT HIV-1 infectivity observed in cells expressing CPSF6 chimeras are dependent on CA-CPSF6 binding (Fig 3A).
(A) Infection with HIV-1 CA mutant N74D luciferase-reporter virus in growth arrested HeLa cells reveals a dependence on CA-CPSF6 binding for the observed changes in WT HIV-1 infectivity. (B) Infection with P90A CA mutant in growth arrested HeLa cells results in less-severe infectivity defect with CPSF6-NP NLS chimera cells, while CPSF6-HNRNP K NLS cells trend to be more sensitive to infection relative to WT virus and C-MYC NLS cells only promote increased infection with WT HIV-1 and not P90A. Relative infectivity in CsA-treated growth arrested cells to untreated WT HIV-1-infected (C) and P90A infected (D) growth arrested cells displays a decrease to no change in infectivity for all CPSF6-NLS cell lines except for CPSF6-NP NLS cells during WT HIV-1 infection, which display an almost twofold increased infection. (E) Infection with non-HIV-1 primate lentiviral luciferase reporter viruses, HIV-2 (Red) and SIVmac (Purple), in growth arrested cells. Relative to HIV-1 infection (Green), there are decreases in HIV-2 infectivity in SV40 and HNRNP K cells, whereas SIVmac appears fairly similar to HIV-1. Results (mean ± SEM) are representative from 3 independent experiments with at least technical duplicate samples per experiment. Statistical analysis was determined using two-way ANOVA. Significant differences are indicated: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. CsA–Cyclosporine A. CPSF6-FL–Full Length CPSF6.
The P90A HIV-1 CA mutant is defective in its ability to bind CypA [56–58], which is another host factor implicated in HIV-1 nuclear import [59]. Importantly, P90A retains the ability to bind to CPSF6, but has been shown to utilize a nuclear import pathway distinct from WT HIV-1 [16]. To further explore the role of the CA-CPSF6 interaction in HIV-1 infection, we infected our cell lines with the P90A mutant virus. Broadly, CPSF6 chimeras that were able to rescue infection by WT HIV-1 were also able to rescue P90A infection to a similar degree, relative to the CPSF6-FL complemented cell line (Fig 3B). However, the C-MYC NLS supported less efficient P90A infection relative to WT HIV-1 infection, while the opposite pattern was observed in cells expressing the CPSF6-HNRNP K chimera (Fig 3B). Additionally, we observed that cells expressing the CPSF6-NP chimera were more readily infectable with P90A virus, despite inhibiting WT HIV-1 infection (Fig 3B). To corroborate this observed difference, we infected our cell lines with WT HIV-1 in the presence of cyclosporin A (CsA). CsA blocks CypA binding to CA and thus displays a phenotype similar to the P90A mutation [56–58]. We found that CsA treatment of WT HIV-1 infected CPSF6-NP chimeric cells resulted in significantly increased infectivity relative to untreated cells, while other chimeric CPSF6-NLS cell lines displayed no change or decreased infectivity following CsA treatment (Fig 3C). Notably, CsA treatment of P90A infected cells resulted in little to no effect on infection in all cell lines tested, confirming that the observed changes were specific to the CA-cyclophilin interaction and not off-target CsA effects (Fig 3D). This demonstrates that disruption of CA-CypA binding can modulate the ability of some CPSF6-NLS chimeras to productively support HIV-1 infection.
Binding to CPSF6 is a shared characteristic amongst primate lentiviruses [35]. We therefore wondered how CPSF6-NLS chimera expression would impact infection with HIV-2 and simian immunodeficiency virus from macaque (SIVmac). In general, HIV-2 and SIVmac were similarly influenced by the expression of CPSF6-NLS chimeras as WT HIV-1 (Fig 3E). However, some differences were noted. HIV-2 infection was relatively less efficient in CPSF6-SV40 NLS and CPSF6-HNRNP K NLS expressing cells, compared to HIV-1 and SIVmac (Fig 3E). Cells expressing the CPSF6-Rac3 NLS were less supportive of both HIV-2 and SIVmac expression, and SIVmac infection did not exhibit the same increase in infectivity as observed with HIV-1 and HIV-2 following infection of CPSF6-C-Myc NLS expressing cells (Fig 3E). These results show that the ability of CPSF6-NLS constructs to facilitate infection was generally conserved among primate lentiviruses that utilize CPSF6 as a host cofactor.
CPSF6-NLS chimeras display cell-type specific phenotypes
To test the CPSF6-NLS chimera constructs in more physiologically relevant settings, we depleted endogenous CPSF6 from T cell (SupT1) and monocytic (THP-1) cell lines and generated stable cells expressing four representative CPSF6-NLS chimeras that were exclusively nuclear in HeLa cells (S1A and S1B Fig). To determine if there were cell-type differences in construct localization, the SupT1 cells and differentiated THP-1 cells were analyzed by confocal microscopy, which revealed chimera CPSF6-NLS construct nuclear localizations similar to those observed in HeLa cells (S1C and S1D Fig). In general, the ability of these CPSF6-NLS chimeras to facilitate HIV-1 infection in SupT1 and THP-1 cells was similar to what was observed in HeLa cells (Fig 4). CPSF6-NP NLS and MX2 NLS constructs in T cells and macrophages behaved largely similarly as they did in HeLa cells (Fig 4). However, similar to the case of capsid mutations and other primate lentiviruses, there were some differences in the ability of some chimeras to promote infection in each cell line, most notably in T cells. In T cells, the CPSF6-SV40 NLS chimera rescued ~50% of infection (Fig 4), whereas it fully rescued infection of differentiated THP-1 cells (Fig 4). Additionally, CPSF6-C-MYC NLS expression increased THP-1 infectivity to about 200% relative to full length CPSF6 (Fig 4), but rescued T cell infectivity to about 100% (Fig 4). Together, these results reveal the target cell can impact the ability of HIV-1 to utilize chimeric CPSF6-NLS constructs during infection.
CPSF6-KO SupT1 T-cells (yellow) and THP-1 monocytes (cyan) were stably transduced with dox-inducible CPSF6-NLS constructs. T cells and HeLa cells were growth arrested prior to infection and THP-1’s were terminally differentiated into macrophages. WT HIV-1 infection was determined by luciferase reporter gene expression. Notably, T cells expressing CPSF6-SV40 NLS were significantly less infectable than corresponding HeLa cells, while CPSF6-SV40 NLS-expressing THP-1 cells supported 100% infection. While CPSF6-C-MYC NLS strongly increased infectivity in HeLa and THP-1 cells relative to CPSF6-FL, this construct conveyed the CPSF6-FL level of infection in T cells. NP and MX2 cells resulted in defective infection in all cell types. Results (mean ± SEM) are representative from 3 independent experiments with at least technical duplicate samples per experiment. Statistical analysis was determined using one-way ANOVA. Significant differences are indicated: ** P<0.01, *** P<0.001, **** P<0.0001.
Heterologous NLSs significantly impact intranuclear stages of HIV-1 infection
Due to the CPSF6-NLS chimera constructs restoring nuclear localization of CPSF6-358 (Fig 1C), we next asked if defective HIV-1 gene expression was a result of defective nuclear import in these cells. HIV-1 nuclear import is commonly quantified by measuring the accumulation of 2-long terminal repeat (LTR)-containing circles, which form via host non-homologous end-joining machinery acting on unintegrated HIV-1 genomes [60]. We infected the chimeric NLS HeLa cell lines with WT HIV-1 and measured reverse transcription (RT; minus-strand after initial strand transfer) and 2-LTR circles by qPCR. All tested chimeric NLS cell lines supported similar levels of HIV-1 DNA synthesis (Fig 5A). CPSF6-358 expression, relative to full-length CPSF6, significantly reduced 2-LTR circle formation (Fig 5B), while CPSF6-KO cells supported nuclear import of viral DNA, as measured by 2-LTR circle formation, consistent with previous studies [26,38]. Interestingly, all four CPSF6-NLS chimeric cell lines examined supported similar levels of 2-LTR circle accumulation (Fig 5B), despite the reduced levels of gene expression observed in CPSF6-358 NP NLS and MX2 NLS-expressing cells (Fig 2A). As integration can impact the amount of viral genomes available to be circularized by nonhomologous end-joining machinery, we repeated infections in the presence of the integrase inhibitor raltegravir (RAL) to block viral DNA integration. When comparing 2-LTR circle formation of RAL treated cells relative to untreated cells, we found no significant differences in nuclear import as measured by 2-LTR circles (S2 Fig). To further examine the ability of these constructs to mediate nuclear import of the vRNPC, we utilized fluorescently tagged Gag-Integrase Ruby (GIR) virus to track the movement of individual vRNPCs (Fig 5C). Using IMARIS software, we determined the percentage of our labeled particles within the nucleus by creating surface masks around the Hoecsht signal to define the nuclear boundary. Consistent with our 2-LTR circle measurements, the CPSF6-NLS chimeric cell lines supported similar levels of nuclear localization of GIR-labelled vRNPCs (Fig 5C and 5D). These results indicate that HIV-1 efficiently entered into the nuclei of our chimeric CPSF6-NLS cell lines and suggests changes in post-nuclear entry steps of infection underlie the differences observed between different CPSF6-NLS chimeras.
(A) RT was measured via amplification of firefly luciferase sequences within the viral genome. (B) 2-LTR circles were measured and normalized to levels obtained in CPSF6-FL-expressing cells. qPCR analysis was performed on genomic DNA isolated from WT HIV-1 infected growth arrested HeLa cells. Gag-Integrase Ruby (GIR) labeled HIV-1 viral particles were used to infect growth-arrested HeLa cell lines. (C) Representative images of GIR-infected HeLa cells 8 h post infection, with Hoechst (blue), GIR (yellow), and SC35 (red). Nuclear virus was determined using IMARIS software and creating a mask around the nuclear Hoechst stain, then determining percent of total GIR particles within the mask. Bottom three rows display inset from top images. (D) Percentage of nuclear GIR signals across the indicated cell lines, which highlighted lack of significant differences. (E) Nuclear penetration was determined via IMARIS software, measuring the distance of nuclear viral particles to the nearest Hoechst edge. GIR in CPSF6-KO cells remained nuclear envelope proximal, whereas other CPSF6 chimeras facilitated nuclear invasion. (F) NS colocalization was determined via GIR infection and subsequent SC35 staining. Percentage of nuclear GIR colocalizing with SC35 was determined using IMARIS software. HIV-1 in CPSF6-NP NLS, CPSF6-MX2 NLS, and CPSF6-KO cells displayed decreased NS colocalization. (G) Representative images showing CPSF6-FL distribution in uninfected versus HIV-1 infected cells, with Hoechst (blue), CPSF6 (green), and SC35 (red). (H) CPSF6 condensation was determined via HA-tag staining at 24 h post-infection. Masks were made around SC35 stain using IMARIS and fluorescent intensity of CPSF6 construct stain within SC35 masks was determined. CPSF6-FL supported much brighter spots consistent with condensation, whereas CPSF6 distribution remained comparable to uninfected cells across other cell lines. All imaging experiments were performed on growth arrested cells. Results in A,B,D and F (mean ± SEM) are representative from 3 independent experiments with at least technical duplicates. Results in E show median+95% CI. Results in H show box and whiskers from min to max. At least 10 images were taken for each condition across 3 independent experiments. Statistical analysis in A,B,D and F was determined using one-way ANOVA and in E and H using Kruskal-Wallis test. Significant differences are indicated: ** P<0.01, *** P<0.001, **** P<0.0001. GIR–Gag-Integrase Ruby. CPSF6-FL–Full-length CPSF6.
Following HIV-1 nuclear import, HIV-1 traffics away from the nuclear envelope and further into the nucleus [30,61]. This inward trafficking step is licensed by CPSF6, as HIV-1 does not efficiently traffic distal from the nuclear envelope in CPSF6-depleted cells [30,61,62]. We quantified viral nuclear penetration by measuring GIR distance to the nearest Hoechst edge at 8 h post-infection. As expected, HIV-1 in CPSF6-KO cells remained near the nuclear periphery (Fig 5E). Viruses in CPSF6-NLS chimera expressing cells, however, penetrated deeper into the nucleus, comparably to cells expressing full-length CPSF6 (Fig 5E).
The CA-CPSF6 interaction facilitates intranuclear trafficking of vRNPCs to NSs [32,33,35,63,64]. To determine if chimeric CPSF6-NLS constructs impair HIV-1 intranuclear trafficking to NSs, we utilized GIR virus in combination with an SC35 stain to visualize colocalization with NSs at 8 h post-infection (Fig 5C and 5F). CPSF6-KO cells displayed a reduction in GIR colocalization with NSs, as described previously using KO cells and/or CA mutant viruses [32–34,63]. However, the SV40 and C-MYC NLS constructs effectively facilitated localization to NSs (Fig 5C and 5F). Alternatively, the NP and MX2 NLS constructs conferred significant decreases in HIV-1 colocalization with NSs (Fig 5F). During infection, CA induces the formation of CPSF6 biomolecular condensates, which colocalize with NSs [63–65]. Due to the SV40-NLS and C-MYC NLS chimeras facilitating viral localization to NSs, we wondered if these mutants could induce the formation of biomolecular condensates, as previously described for full-length CPSF6 [63–65]. By measuring CPSF6 signal intensity colocalizing with SC35, we found that only full-length CPSF6 condensed with NSs, whereas the CPSF6-NLS chimeras retained diffuse nuclear localization similar to uninfected cells (Fig 5G and 5H). Additionally, we determined that there were no differences in the number of nuclear speckles per cell or the total volume of nuclear speckles per cell relative to uninfected cells (S3A and S3B Fig). These results were consistent with previously published findings [33]. Taken together, our results show that a subset of our CPSF6-NLS constructs that supported maximal or partial HIV-1 infection nevertheless supported equivalent levels of HIV-1 nuclear entry. Moreover, following nuclear entry, the SV40 NLS and C-MYC NLS constructs facilitated HIV-1 nuclear trafficking and NS localization despite a lack of biomolecular condensation. Conversely, the NP and MX2 constructs, despite facilitating nuclear penetration, were unable to facilitate NS localization or biomolecular condensation of CPSF6.
CPSF6-NP and MX2 NLS chimeras reduce HIV-1 integration and influence integration site selection
The substrate for HIV-1 integration is the linear viral DNA made by RT. HIV-1 integration favors active chromatin including DNA in close proximity to NSs, called speckle-associated domains (SPADs) [33,35]. We asked if the observed defects in NS colocalization might correlate with changes in overall levels of HIV-1 integration as well as sites of integration within the human genome. To detect total levels of HIV-1 integration, we utilized Alu-Gag qPCR, which amplifies sequences that lay between the HIV-1 gag gene and genomic Alu-repeats; a second, nested qPCR was used to quantify the extent of first round reaction products [66,67]. We observed a significant reduction in integration in CPSF6-358 expressing cells, as expected, due to viral restriction in the cytoplasm (Fig 6A). Interestingly, we also observed a reduction in Alu-Gag products in cells expressing CPSF6-NP NLS and MX2 NLS (Fig 6A). CPSF6-SV40 NLS and C-MYC NLS by contrast supported Alu-Gag integration levels similarly as full-length CPSF6 (Fig 6A). These data are consistent with the notion that overall integration levels largely dictate levels of HIV-1 infection as assessed by viral reporter gene expression (compare Fig 2A and 2C with Fig 6A). To further interrogate the relationship between integration level and viral gene expression, we utilized a dual fluorescent reporter HIV, called HIVGKO [68]. This virus contains a constitutive reporter gene (mKO2) driven by an internal promoter and a second GFP reporter under the control of the viral LTR promoter. We observed indistinguishable levels of expression of both reporter genes across our different cell types, further linking the relationship between overall levels of HIV-1 integration and viral gene expression (S4 Fig).
(A) Alu-Gag qPCR detection of HIV-1 integration levels in growth arrested cells. CPSF6-NP NLS and CPSF6-MX2 NLS cells supported significantly reduced levels of HIV-1 integration. (B-E) HIV-1 integration in growth arrested HeLa cells into defined genomic annotations including genes (B), SPADs (C), LADs (D) and gene-dense regions (E). Integration into genes in CPSF6-C-MYC NLS, CPSF6-NP NLS, and CPSF6-MX2 NLS-expressing cells was reduced compared to cells expressing CPSF6-FL and CPSF6-SV40 NLS. Fractional LAD targeting in CPSF6-C-MYC, NLS CPSF6-NP NLS, and CPSF6-MX2 NLS-expressing cells was increased significantly compared to cells expressing CPSF6-FL and CPSF6-SV40 NLS. Integration in gene dense regions in CPSF6-NP NLS cells was reduced significantly compared to CPSF6-FL expressing cells. Integration in NLS CPSF6-NP NLS and CPSF6-MX2 NLS cells was interestingly statistically indistinguishable from the RIC value. Statistical analysis was determined using one-way ANOVA in A and chi-squared test followed by students t-test in B-E. * P<0.05, *** P<0.001, **** P<0.0001 (black asterisks, vs CPSF6-FL cells; grey asterisks, vs corresponding RIC).
To further probe the granularity of the integration phenotypes, we mapped sites of HIV-1 integration within the human genome essentially as described previously [69,70]. In short, asymmetric linkers were ligated onto sheared genomic DNA fragments, and LTR-linker sequences amplified via semi-nested PCR were analyzed by Illumina sequencing. Unique sites of HIV-1 integration were then mapped with respect to several genomic features including genes, SPADs, lamina associated domains (LADs), alphoid repeat elements, LINE1 repeat elements, and gene dense regions [30,33,71–75]. Computationally-generated random integration control (RIC) values were also included for each genomic annotation.
The number of unique integration sites mapped from a total of 2 to 4 independent infection experiments across cell lines varied from 925 to >10,000 (S1 Table). The comparatively low number of sites recovered from MX2 NLS and NP NLS-expressing cells– 925 and 1,125, respectively–was, on the one hand, consistent with the comparatively low level of overall integration events detected by Alu-Gag PCR (Fig 6A). On the other hand, due to these comparatively low numbers, concern was raised as to whether the number of sites recovered could support meaningful conclusions regarding HIV-1 integration site targeting. Importantly, prior studies that analyzed much fewer sites of HIV-1 integration in the human genome, including 524 sites [76], 84 to 222 sites [77], or 110 to 1,485 sites [78], were able to reach meaningful conclusions. The ability to reach meaningful conclusions regarding integration targeting additionally relies on the degree of overlap between mapped integration sites and the genomic annotations of interest. The observed degree of overlap is itself dependent in part on the completeness of a given annotation-set for a given reference genome. To address these issues head-on, we conducted a power analysis for a chi-squared test assuming a power of (1-β) = 0.8 and a two-sided significance threshold of α = 0.05 to assess the relevance of MX2 NLS and NL NLS datasets for downstream analyses. The power analysis revealed that for gene, gene dense regions, and SPAD overlaps, there was sufficient depth across datasets to make conclusions with respect to CPSF6-FL and RIC at the stated power. For LAD overlaps, all comparisons except the MX2-RIC comparison met the stated power threshold. The MX2-RIC comparison had a calculated power of 0.75, which was reasonably close to the posited 0.8 value. Integration site overlap comparisons with LINE1 repeat elements showed sufficient power with respect to RIC, but were slightly underpowered with respect to CPSF6-FL (calculated power values of 0.76 for NP NLS and 0.71 for MX2 NLS), so we will restrict interpretations of LINE1 datasets to the RIC comparator. HIV-1 integration avoids alphoid repeats [73] and alphoid repeat coordinates are relatively poorly annotated for human genome build hg19. Accordingly, quantified integration site overlaps with alphoid repeats were insufficient to draw any meaningful conclusions, and thus integration targeting of alphoid repeats will not be commented on further (S3 Table).
As expected, integration into genes in CPSF6-FL-expressing cells was significantly enriched compared to the RIC value (Fig 6B, grey asterisks). While genic integration in CPSF6-358 SV40 NLS-expressing cells was statistically indistinguishable from CPSF6-FL cells, ~2–7% reductions in integration into genes was observed in C-MYC, NP, and MX2 NLS-expressing cells (Fig 6B and S1 Table). Overall similar trends were observed for integration into SPADs, LADs, and gene dense regions. Thus, while SPAD-proximal integration was marginally reduced by ~1.7% in SV40 NLS-expressing cells (P = 0.03), SPAD targeting was reduced by ~5.4% to 8.7% in C-MYC, NP, and MX2 NLS-expressing cells (P<0.0001) (Fig 6C and S2 Table). Reciprocal upticks in integration into LADs in C-MYC, NP, and MX2 NLS-expressing cells differed significantly from LAD targeting values observed in CPSF6-FL and SV40-expressing cells (P<0.0001) (Fig 6D). In terms of gene dense regions, the 3.75 genes/Mb reduction in NP NLS-expressing cells was statistically significant versus the 15.4 genes/Mb value observed in CPSF6-FL cells. Interestingly, integration into gene dense regions in NP and MX2 NLS-expressing cells, compared to CPSF6-FL containing cells, was no longer enriched compared to the RIC 9.2 genes/Mb value (Fig 6E and S2 Table).
The SV40 NLS chimera behaved most similar to CPSF6-FL with respect to integration into genes, SPADs, and LADs, showing no or comparatively marginal differences across these annotations. The C-MYC, NP, and MX2 NLSs generally showed increasingly significant differences with respect to CPSF6-FL overlap with genes, SPADs, and LADs. Notably, CPSF6-FL and SV40 NLS showed indistinguishable overlap with LINE1 repeat elements relative to RIC. C-MYC, NP, and MX2 NLSs, conversely, showed slight increases in LINE1 integration relative to RIC (S2 Table). These apparent differences, however, were comparatively small with marginal statistical significance (P = 0.008 to 0.03). Altogether, our results revealed that the NP and MX2 constructs, which were defective in supporting overall levels of HIV-1 integration (Fig 6A), supported marginally less, though statistically significant, integration into genomic annotations associated with active chromatin, including genes and SPADs (Fig 6B and 6C). Collectively, our results establish the importance of CPSF6’s NLS in facilitating the intranuclear events of HIV-1 infection.
Discussion
In this study, we determined that the NLS utilized by CPSF6 can impact various stages of nuclear trafficking and integration. By attaching heterologous NLSs to a truncated CPSF6, CPSF6-358, we observed a range of outcomes that were dependent on the NLS utilized. In line with previous experiments that utilized MX2 as a model system, some NLSs failed to complement the nuclear localization defect of CPSF6-358, and as a result these chimeric proteins restricted HIV-1 infection in the cytoplasm [23,41]. This is generally consistent with the ability of different nuclear import pathways, perhaps through heterogenous NPCs, to accommodate different nuclear transport cargoes [23,40]. We hypothesize that multiple NPC subsets can be found within a cell, and these NPC subsets have alternative nuclear transport mechanisms [23].
Following infection with HIV-1, we observed differences in reporter gene expression amongst the nuclear-rescued CPSF6-358 constructs. Some of the NLSs (C-MYC, Rac3) displayed a slight, but significant increase in reporter gene expression relative to full-length CPSF6. Additionally, the C-MYC NLS construct resulted in marginally increased levels of integration as measured by Alu-Gag qPCR assays. Although omitted from downstream analyses, the Rac3 NLS protein behaved similar to the C-MYC protein in HIV-1 infection assays. Several additional constructs, including SV40, HNRNP K, and HNRNP A1, supported WT HIV-1 infection levels similar to CPSF6-FL.
Our results reveal that infection of CPSF6-NP and MX2 NLS cells resulted in functional nuclear import, as measured by both 2-LTR circles and localization of Gag-Integrase labeled viruses inside the nucleus of these infected cells. However, we found that infection in these cell lines resulted in decreased levels of viral integration as measured by Alu-Gag qPCR. The observation that 2-LTR circle formation was similar in cells with nuclear localized CPSF6 fusions suggests that the fusion constructs do not negatively impact core uncoating during infection, as 2-LTR circle formation requires the non-homologous end joining machinery. Additionally, it is known that abolishing the CA/CPSF6 interaction alters the spatiotemporal staging of infection, such that the viral core remains associated with the nuclear envelope [31,61,62]. However, we observed nuclear penetration of vRNPCs in cells expressing CPSF6-NLS fusions, regardless of their ability to facilitate gene expression (Fig 5E). Although we did not specifically interrogate the levels of CA associated with vRNPCs in these studies, the degree to which nuclear penetration is known to be mediated by CPSF6/CA interactions suggests that CA remains associated with the vRNPC in cells expressing CPSF6-NLS fusions.
Our integration site analyses revealed that HIV-1 in CPSF6-NP and MX2 NLS-expressing cells generally disfavored regions associated with active chromatin, such as genes and SPADs, which could potentially further reduce the levels of gene expression supported by these integrated proviruses. We speculate that the overall reduced levels of integration observed in CPSF6-NP and MX2 NLS cells combined with the propensity for these proviruses to disfavor active chromatin account for the comparatively poor expression profiles of these proviruses. Of note, our work fails to address why overall integration is significantly diminished in CPSF6-NP and MX2 NLS-expressing cells. Possibly, HIV-1 encounters a novel nuclear restriction factor(s) via the altered paths of nuclear import instilled by these NLSs or regions that have tightly packed chromatin compared to the regions that have loose or relaxed structures associated with active genes. Further work will need to be done to explore these hypotheses.
Prior studies have investigated the differences in the nuclear import pathways of WT HIV-1 and capsid mutant viruses P90A [16,39] and N74D [26], showing that binding to CypA/Nup153 and CPSF6, respectively, significantly impacted nuclear import pathway utilization. In our studies, the N74D CA mutant, which is defective for CPSF6 binding, predictably infected all CPSF6 chimeric construct expressing cells equally. However, infection with the P90A mutant resulted in differential infectivity relative to the WT virus. Specifically, P90A was less sensitive than WT HIV-1 to restriction in CPSF6-NP NLS cells, which was reproduceable following CsA treatment. Similarly, P90A displayed greater relative infectivity than WT HIV-1 in CPSF6-HNRNP K NLS cells, highlighting a potential role of cyclophilin binding influencing sensitivity in these cell lines. We speculate this may be due to P90A virus entering the nucleus via an alternative nuclear import pathway, as we and others have shown [16,39,40]. In this case, perhaps the activity of the CPSF6-NLS effects in the nucleus is modulated by the nuclear import pathway utilized.
We also observed differential sensitivities to the CPSF6-NLS chimeras amongst related lentiviruses, as infection with HIV-2 and SIVmac highlighted further virus-specific phenotypes. Additionally, CPSF6-NLS chimeras displayed differential phenotypes, specifically in the SV40 and C-MYC NLS chimeras, in T cell and macrophage cell lines, where we observed SV40-NLS only partially rescued infection in T cells but fully rescued infection in macrophages and C-MYC-NLS rescued infection to WT levels in T cells but doubled infectivity in macrophages.
Previous reports have shown that the CPSF6 C-terminal RSLD is an IDR that can phase separate as a GFP-RSLD fusion protein in vitro [43]. Moreover, purified mCherry-CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro [79]. These observations raise the possibility that the RSLD plays a role in CPSF6 biomolecular condensate formation during HIV-1 infection [43,63,65], which we have recently confirmed [80]. The results reported here are fully consistent with this notion, as we found that only full-length CPSF6, which contains an intact RSLD, formed CPSF6 condensates following HIV-1 infection, while the CPSF6-358 constructs lacking an RSLD remained pan-nuclear in distribution. Interestingly, infection in cell lines expressing CPSF6-SV40 NLS and CPSF6-C-MYC NLS constructs retained HIV-1 NS colocalization. This was a surprising finding, suggesting that HIV-1 NS colocalization can occur independent of CPSF6 LLPS activity, as the constructs containing the SV40 and C-MYC NLS effectively trafficked HIV-1 to NSs despite lacking the RSLD.
Collectively, our results reveal that NLSs have roles beyond targeting cargo for nuclear import, such as impacting viral subnuclear localization following nuclear entry. To our knowledge, there is little precedent for post-nuclear entry roles for NLSs. As such, our results utilizing HIV-1 infection as a readout for the nuclear function of NLSs provide a template to understand the nuclear trafficking or other post-nuclear activities that may be imparted by specific NLSs.
Materials and methods
Cell lines
THP-1 and SupT1 cells were obtained from ATCC. 293T and HeLa cells were cultured in DMEM and THP-1 and SupT1 cells were cultured in RPMI. Both mediums were supplemented with 10% tetracycline-depleted fetal calf serum (R&D Systems), 1000 U/ml of penicillin, and 1000 U/ml of streptomycin. THP-1 cells were differentiated into macrophages with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 48 h, followed by 48 h rest with normal media without PMA before experiments.
Constructs
Truncated CPSF6 containing amino acids 1–358 (CPSF6-358) from human isoform 1 was cloned into the pCW57.1 backbone (Addgene 71782). A hemagglutinin (HA) tag as well as heterologous NLSs (Table 1) were appended onto the C-terminus of CPSF6-358. These constructs were under a doxycycline inducible promoter, and expression was induced with 5 μg/ml doxycycline for 48 h before viral infection.
Virus and vector production
To generate viral vectors containing CPSF6-NLS constructs, 293T cells were seeded in 10 cm dishes and co-transfected with 4 μg pCW57-CPSF6-NLS, 4 μg psPAX packaging plasmid, and 2 μg pCMV-VSVg with polyethylenimine overnight. Media was changed the next morning and pseudotyped vector was collected 48 h post transfection. To generate pseudotyped HIV-1, HIV-2, SIVmac, N74D, and P90A viruses, 293T cells were seeded in 15 cm plates and transfected with 20 μg WT/N74D/P90A NL4.3 containing LTR driven luciferase and mCherry reporter genes (separated by a T2A ribosomal skip sequence), HIVGKO, HIV-2-Luciferase, or SIVmac-Luciferase [81] constructs with 5 μg pCMV-VSVg using polyethylenimine. The HIV-2-Luciferase construct was made by swapping the GFP gene of HIV-2-GFP for the firefly luciferase gene, as previously described for analogous equine infectious anemia constructs [35]. Media was changed the following morning and virus was collected at 48 and 72 h post transfection. NL4.3-based plasmid pNLXLuc.R-U3-tag [70] was used to make virus for integration site distribution experiments. To generate Gag-integrase-Ruby viral particles, 293T cells were seeded in 10 cm plates and transfected with 4.5 μg R7ΔEnv, 3.5 μg Gag-integrase-Ruby, and 2 μg pCMV-VSVg using polyethylenimine. Viral particles were collected 48 h post transfection. All viruses and vectors were filtered through 0.45-μm filters (Merck Millipore). Virus was concentrated by spinning at 5000 g overnight at 4°C. Reverse transcriptase units of viral preps were determined using SG-PERT assay as previously described [82]. Viral infections were synchronized by spinoculation at 1200 g for 2 h at 13°C.
Stable cell line generation
CPSF6-knockout cell lines were generated using pLentiCRISPRv2 with previously described guides [83] under blasticidin selection. For HeLa cells, a concentration of 5 μg/ml blasticidin was used and for THP-1 and SupT1 cells, a concentration of 2 μg/ml was used. Following HeLa cell selection, cells were sorted by flow cytometry and single-cell clonal populations were verified by western blot. CPSF6 knockout cells were transduced with viral vectors containing CPSF6-NLS constructs under puromycin selection. 5 μg/ml puromycin was used for selection of HeLa and 2 μg/ml puromycin was used for THP-1 and SupT1 cells. Construct expression was verified by western blot after 48 h doxycycline induction.
Antibodies and chemicals
HIV-1 capsid, p24, was stained using mouse monoclonal anti-HIV-1 p24 from Santa Cruz Biotechnology (catalogue no. sc-69728). SC35 was stained using mouse monoclonal anti p-SC35 from Santa Cruz Biotechnology (catalogue no. sc-53518). GAPDH was stained with mouse monoclonal anti GAPDH from Santa Cruz Biotechnology (catalogue no. sc-47724). CPSF6 was stained using either CPSF6 polyclonal antibody from Invitrogen (catalogue no. PA5-41830) or from Abcam (catalogue no. ab175237). HA-tag was stained using either rabbit anti-HA-tag from Sigma-Aldrich (catalogue no. H6908) or mouse anti-HA-Tag (6E2) from Cell Signaling Technology (catalogue no. 2376). Secondary antibodies with conjugated fluorophores used in immunofluorescence experiments were purchased from Jackson Immunoresearch Laboratories. Aphidicolin (Cayman Chemicals, catalogue no. 14007) was used to growth arrest cells. Doxycycline hyclate (Sigma-Aldrich, catalogue no. D9891) was used to induce construct expression. CsA was purchased from Sigma-Aldrich. RAL was purchased from MedChemExpress (Catalogue no. HY-10353). Hoechst was purchased from ImmunoChemistry Technologies (Catalogue no. 639).
Western blot
Cell lysates were prepared using NP-40 lysis buffer (100 mM Tris pH 8.0, 1% NP-40, 150 mM NaCl) containing protease inhibitor cocktail (Roche) for 20 min with shaking on ice. Lysates were then centrifuged at 16000 g for 2 min and supernatant was collected. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS was then added to lysates and incubated at 95°C for 5 min. Equal amounts of total protein were loaded into a 4–15% gradient gel (Bio-Rad Laboratories). Proteins following electrophoresis were then transferred to a nitrocellulose membrane (Bio-Rad Laboratories). Nitrocellulose membranes were incubated with specific primary antibodies overnight at 4°C on a rocker. Secondary antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific) were added for 1 h at room temperature on a rocker. Chemiluminescence was detected using ProteinSimple imaging system (FluorChem E).
Quantitative PCR (qPCR)
Cells were infected with equal reverse transcriptase units as determined by SG-PERT assay. qPCR was used to determine reverse transcription, 2-LTR circle formation, and integration. DNA was isolated from cells using DNeasy Blood and Tissue Kit protocol (QIAGEN). DNA concentration was determined using NanoDrop 2000 spectrophotometer and diluted to equal concentrations. The following primers were used for 2-LTR circle qPCRs: 2-LTR Forward, 5’-AACTAGGGAACCCACTGCTTAAG-3’; 2-LTR Reverse, 5’-TCCACAGATCAAGGATATCTTGTC-3’ [84]. Primers used to measure reverse transcription were specific for the Firefly Luciferase gene present in our virus. Firefly Luc Fwd: 5’-CGGAAAGACGATGACGGAAA-3’. Firefly Luc Rev: 5’-CGGTACTTCGTCCACAAACA-3’. Human beta actin was used as housekeeping gene for normalization: HuBeta Actin Fwd, 5’-TCACCCACACTGTGCCCATCT-3’; HuBeta Actin Rev, 5’-CAGCGGAACCGCTCATTGCCAATGG-3’.
The Alu-gag PCR assay for HIV-1 integration was performed as described previously [67,74,85]. Due to lentiviral transduction to generate cell lines, modifications were made to primer sets used. Lambda T sequence was added to the reverse primer of reaction 1 to add specificity and reduce background for reaction 2. Primers used for reaction 1 were as follows. Alu Forward: 5’-GCCTCCCAAAGTGCTGGGATTACAG-3’. Gag-LambdaT Reverse: 5’-AGTTTCGCTTACGTGGCATGTTCCTGCTATGTCACTTCC-3’. Second reaction primers for qPCR were as follows. HIV-1 Gag Forward: 5’-TCAGCCCAGAAGTAATAC-3’. Lambda T Reverse: 5’-AGTTTCGCTTACGTGGCAT-3’.
Infectivity assays
Cells were plated at equal densities and growth arrested (where indicated) in 5 μg/ml aphidicolin overnight. Infection was synchronized as described above. Infectivity was measured at 48 h post infection by either luciferase assay or flow cytometry. Following infection with luciferase reporter viruses, cells were lysed in Passive Lysis Buffer (Promega) and luciferase activity was measured using a luminometer (Promega, GloMax Navigator). To monitor infection from the mCherry reporter, the percent of mCherry positive cells were determined using flow cytometry (BD Bioscience, BD FACSCanto II cytometer).
Microscopy
Images were acquired using a DeltaVision widefield fluorescence microscope (Applied Precision) with a digital camera (CoolSNAP HQ; Photometrics) and 1.4-numerical aperture 100X objective lens. Excitatory light was created by Insight SSI solid-state illumination module (Applied Precision). Images were acquired in several Z-stacks and deconvolution was performed using the softWoRx software v.7.0.0 (Applied Precision, GE Healthcare). Deconvolved images were analyzed using Imaris x64 v.7.6.4 (Bitplane). The surfaces or spots function of Imaris was used to analyze the signal of interest. The same algorithm was used for all images of an experiment. ImageJ was used to determine raw integrated density to quantify percentage of staining within the nucleus.
Statistical analysis
Prism 10.0 (GraphPad) was used to generate graphs and for statistical analysis. Statistical significance was determined using a one-way or two-way ANOVA analysis, where multiple comparisons were measured against full-length CPSF6 (CPSF6-FL). Data are graphed as mean ± standard error mean, except when indicated otherwise in figure legend. Kruskal-Wallis test was performed where indicated in figure legends. Significant differences are indicated: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Integration site analysis
Infections were synchronously performed with U3-modified HIV-1 [70] at ~1 MOI to enable LTR-specific PCR amplification in cells containing pre-existing CPSF6-358 NLS lentiviral vectors. Genomic DNA was isolated from infected cells 72 h post infection using Quick-DNA Miniprep kit (Zymo Research, catalogue no. D3024).
Integration site sequencing libraries were prepared essentially as described previously [69,70]. Briefly, 5 μg genomic DNA was digested overnight with a restriction enzyme cocktail containing 100 U each of NheI-HF, AvrII, SpeI-HF, and BamHI-HF (New England Biolabs). Digested DNA was purified using a GeneJet PCR purification kit. Freshly annealed asymmetric linkers were subsequently ligated to the fragmented gDNA in four parallel overnight reactions. These reaction products were then purified and subjected to two rounds of ligation mediated (LM)-PCR. The LM-PCR products were then purified and yield was quantified by DNA fluorimetry (Qubit). LM-PCR products were pooled at 10 nM total concentration, ΦX174 control DNA was added to the pooled libraries at 30% molar concentration, and the mixture was loaded onto a P2 300 cycle cartridge at 650 pM concentration for sequencing on an Illumina NextSeq2000 sequencer. Resulting raw FASTQ files were demultiplexed with Sabre (https://github.com/najoshi/sabre), the demultiplexed reads were trimmed to remove HIV-1 LTR and linker sequences, and the trimmed reads were aligned to human genome build hg19 as described [69]. Uniquely mapped integration site annotations were used in downstream analyses. SPAD annotations used here were the same as previously described [33,35]. LAD annotations were downloaded from the 4D Nucleome data portal (Experiment Set 4DNESTAJJM3X).
Random integration control (RIC) datasets were generated by randomly placing integration sites within hg19 in silico. Following integration site placement, the genome was cleaved at the nearest downstream NheI, AvrII, SpeI, or BamHI recognition site. Generated fragments were filtered to be between 14 bp and 1200 bp and paired-end sequencing reads were simulated by taking up to 150 bp from both fragment ends to generate R1 and R2 fasta files. These fasta files were then aligned to hg19 and the location of uniquely mappable random integration sites were obtained. LINE1 and alphoid repeat annotations were obtained from the hg19 RepeatMasker track annotations provided by UCSC (https://genome.ucsc.edu/cgi-bin/hgTrackUi?g=rmsk).
Integration site overlap with genomic features (genes, SPADs, LADs, LINE1 repeats, and alphoid repeats) and gene density within 1 Mb of integration sites were quantified with the GenomicRanges Bioconductor package [86]. For comparison of feature overlap between conditions, IS datasets from 2 or more independent replicates for each condition were pooled and the combined data was used for statistical comparisons. The fraction overlap values and the corresponding standard deviations reported in S1 Table reflect the mean and standard deviation between replicates weighted according to dataset size. Statistical comparisons of feature overlap were made using the χ2 test for equal proportions. Student’s t-test was used to assess statistical significance of gene density around integration sites between conditions.
Supporting information
S1 Fig. Chimeric construct expression and localization in T cells and macrophages.
Western blot analysis of CPSF6-depleted stably-transduced SupT1 (A) and THP-1 (B) cell lines using anti-CPSF6 antibody to detect CPSF6-NLS construct expression following 48 h of doxycycline induction. Anti-GAPDH antibody used as loading control. Immunofluorescent imaging of SupT1 cell lines (C) and THP-1 cell lines differentiated into macrophages (D) using anti-HA antibody. Partial cytoplasmic staining of CPSF6-358 is evident in both cell types (highlighted in SupT1 cells by white arrow).
https://doi.org/10.1371/journal.ppat.1012354.s001
(TIFF)
S2 Fig. 2-LTR circle formation in RAL-treated versus untreated HIV-1 infected cells.
qPCR analysis was performed on genomic DNA isolated from WT HIV-1 infected cells in the presence or absence of raltegravir (RAL). No significant differences in 2-LTR circles were noted among tested cell lines relative to untreated HIV-1 infected cells. Results (mean ± SEM) are representative from 3 independent experiments with at least technical duplicates.
https://doi.org/10.1371/journal.ppat.1012354.s002
(TIFF)
S3 Fig. Nuclear speckle numbers and total volume following infection.
WT HIV-1 infected HeLa cells were fixed 24 h post-infection and stained for the nuclear speckle marker SC35. (A) IMARIS software was used to form surfaces around SC35 staining in the nucleus. Total SC35 surfaces per cell was determined and no significant differences were observed among the tested cell lines relative to uninfected control cells. (B) IMARIS software was utilized to determine the total volume of SC35 surfaces per cell. No significant differences among the tested HIV-1 infected cell lines relative to uninfected control cells were observed. At least 10 images were taken for each condition across 3 independent experiments.
https://doi.org/10.1371/journal.ppat.1012354.s003
(TIFF)
S4 Fig. HIVGKO infection.
HeLa cells infected with dual reporter HIVGKO virus. mKO2 reporter is constitutive and GFP reporter is under control of the HIV LTR promoter. Comparable levels of mKO2 and GFP in the tested cell lines indicates that relative integration levels determined viral gene expression.
https://doi.org/10.1371/journal.ppat.1012354.s004
(TIFF)
S1 Table. Integration distributions in CPSF6-NLS chimera HeLa cellsa.
aResults (weighted mean ± weighted SD) are from ≥ two independent infection experiments. bRIC–random integration control.
https://doi.org/10.1371/journal.ppat.1012354.s005
(DOCX)
S2 Table. Integration site statistics in CPSF6-NLS chimera HeLa cells.
aC6FL–CPSF6-FL. RIC–random integration control.
https://doi.org/10.1371/journal.ppat.1012354.s006
(DOCX)
S3 Table. Power analysis of integration site statisticsa.
aComparisons highlighted in red are underpowered. bC6FL–CPSF6-FL. RIC–random integration control. cRequired sample sizes to achieve Power = 0.8 with Alpha = 0.05 for left (n1) and right (n2) side of comparison as stated in comparison column.
https://doi.org/10.1371/journal.ppat.1012354.s007
(DOCX)
References
- 1. Ori A, Banterle N, Iskar M, Andres-Pons A, Escher C, Khanh Bui H, et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol Syst Biol. 2013;9:648. pmid:23511206
- 2. Ribbeck K, Gorlich D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 2001;20(6):1320–30. pmid:11250898
- 3. Yang W, Gelles J, Musser SM. Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci U S A. 2004;101(35):12887–92. pmid:15306682
- 4. Bonner WM. Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J Cell Biol. 1975;64(2):421–30. pmid:46868
- 5. Paine PL, Moore LC, Horowitz SB. Nuclear envelope permeability. Nature. 1975;254(5496):109–14. pmid:1117994
- 6. Adam SA, Lobl TJ, Mitchell MA, Gerace L. Identification of specific binding proteins for a nuclear location sequence. Nature. 1989;337(6204):276–9. pmid:2911368
- 7. Miyamoto Y, Yamada K, Yoneda Y. Importin alpha: a key molecule in nuclear transport and non-transport functions. J Biochem. 2016;160(2):69–75.
- 8. Pornillos O, Ganser-Pornillos BK, Kelly BN, Hua Y, Whitby FG, Stout CD, et al. X-ray structures of the hexameric building block of the HIV capsid. Cell. 2009;137(7):1282–92. pmid:19523676
- 9. Gres AT, Kirby KA, KewalRamani VN, Tanner JJ, Pornillos O, Sarafianos SG. STRUCTURAL VIROLOGY. X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability. Science. 2015;349(6243):99–103. pmid:26044298
- 10. Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI. Assembly and analysis of conical models for the HIV-1 core. Science. 1999;283(5398):80–3. pmid:9872746
- 11. Mattei S, Glass B, Hagen WJ, Krausslich HG, Briggs JA. The structure and flexibility of conical HIV-1 capsids determined within intact virions. Science. 2016;354(6318):1434–7. pmid:27980210
- 12. Zhao G, Perilla JR, Yufenyuy EL, Meng X, Chen B, Ning J, et al. Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics. Nature. 2013;497(7451):643–6. pmid:23719463
- 13. Yamashita M, Emerman M. Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol. 2004;78(11):5670–8. pmid:15140964
- 14. Bichel K, Price AJ, Schaller T, Towers GJ, Freund SM, James LC. HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358. Retrovirology. 2013;10:81. pmid:23902822
- 15. Fu L, Weiskopf EN, Akkermans O, Swanson NA, Cheng S, Schwartz TU, et al. HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor. Nature. 2024;626(8000):843–51. pmid:38267583
- 16. Schaller T, Ocwieja KE, Rasaiyaah J, Price AJ, Brady TL, Roth SL, et al. HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency. PLoS Pathog. 2011;7(12):e1002439. pmid:22174692
- 17. Bhattacharya A, Alam SL, Fricke T, Zadrozny K, Sedzicki J, Taylor AB, et al. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6. Proc Natl Acad Sci U S A. 2014;111(52):18625–30. pmid:25518861
- 18. Matreyek KA, Yucel SS, Li X, Engelman A. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity. PLoS Pathog. 2013;9(10):e1003693. pmid:24130490
- 19. Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, et al. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly. PLoS Pathog. 2014;10(10):e1004459. pmid:25356722
- 20. Shen Q, Feng Q, Wu C, Xiong Q, Tian T, Yuan S, et al. Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels. Nat Struct Mol Biol. 2023;30(4):425–35. pmid:36807645
- 21. Dickson CF, Hertel S, Tuckwell AJ, Li N, Ruan J, Al-Izzi SC, et al. The HIV capsid mimics karyopherin engagement of FG-nucleoporins. Nature. 2024;626(8000):836–42. pmid:38267582
- 22. Shen Q, Kumari S, Xu C, Jang S, Shi J, Burdick RC, et al. The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores. Proc Natl Acad Sci U S A. 2023;120(13):e2202815120. pmid:36943880
- 23. Kane M, Rebensburg SV, Takata MA, Zang TM, Yamashita M, Kvaratskhelia M, et al. Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2. Elife. 2018;7:e35738. pmid:30084827
- 24. Price AJ, Fletcher AJ, Schaller T, Elliott T, Lee K, KewalRamani VN, et al. CPSF6 defines a conserved capsid interface that modulates HIV-1 replication. PLoS Pathog. 2012;8(8):e1002896. pmid:22956906
- 25. Wei G, Iqbal N, Courouble VV, Francis AC, Singh PK, Hudait A, et al. Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection. Nat Commun. 2022;13(1):5879. pmid:36202818
- 26. Lee K, Ambrose Z, Martin TD, Oztop I, Mulky A, Julias JG, et al. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe. 2010;7(3):221–33. pmid:20227665
- 27. Gruber AR, Martin G, Keller W, Zavolan M. Cleavage factor Im is a key regulator of 3’ UTR length. RNA Biol. 2012;9(12):1405–12. pmid:23187700
- 28. Di Nunzio F, Danckaert A, Fricke T, Perez P, Fernandez J, Perret E, et al. Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration. PLoS One. 2012;7(9):e46037. pmid:23049930
- 29. Zhang R, Mehla R, Chauhan A. Perturbation of host nuclear membrane component RanBP2 impairs the nuclear import of human immunodeficiency virus -1 preintegration complex (DNA). PLoS One. 2010;5(12):e15620. pmid:21179483
- 30. Achuthan V, Perreira JM, Sowd GA, Puray-Chavez M, McDougall WM, Paulucci-Holthauzen A, et al. Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration. Cell Host Microbe. 2018;24(3):392–404 e8. pmid:30173955
- 31. Bejarano DA, Peng K, Laketa V, Borner K, Jost KL, Lucic B, et al. HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex. Elife. 2019;8:e41800. pmid:30672737
- 32. Selyutina A, Persaud M, Lee K, KewalRamani V, Diaz-Griffero F. Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating. Cell Rep. 2020;32(13):108201. pmid:32997983
- 33. Francis AC, Marin M, Singh PK, Achuthan V, Prellberg MJ, Palermino-Rowland K, et al. HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains. Nat Commun. 2020;11(1):3505. pmid:32665593
- 34. Guedan A, Donaldson CD, Caroe ER, Cosnefroy O, Taylor IA, Bishop KN. HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration. PLoS Pathog. 2021;17(9):e1009484. pmid:34543344
- 35. Li W, Singh PK, Sowd GA, Bedwell GJ, Jang S, Achuthan V, et al. CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration. mBio. 2020;11(5):e02254–20. pmid:32994325
- 36. Koh Y, Wu X, Ferris AL, Matreyek KA, Smith SJ, Lee K, et al. Differential effects of human immunodeficiency virus type 1 capsid and cellular factors nucleoporin 153 and LEDGF/p75 on the efficiency and specificity of viral DNA integration. J Virol. 2013;87(1):648–58. pmid:23097450
- 37. Dettwiler S, Aringhieri C, Cardinale S, Keller W, Barabino SM. Distinct sequence motifs within the 68-kDa subunit of cleavage factor Im mediate RNA binding, protein-protein interactions, and subcellular localization. J Biol Chem. 2004;279(34):35788–97. pmid:15169763
- 38. Jang S, Cook NJ, Pye VE, Bedwell GJ, Dudek AM, Singh PK, et al. Differential role for phosphorylation in alternative polyadenylation function versus nuclear import of SR-like protein CPSF6. Nucleic Acids Res. 2019;47(9):4663–83. pmid:30916345
- 39. Matreyek KA, Engelman A. The requirement for nucleoporin NUP153 during human immunodeficiency virus type 1 infection is determined by the viral capsid. J Virol. 2011;85(15):7818–27. pmid:21593146
- 40. Dharan A, Bachmann N, Talley S, Zwikelmaier V, Campbell EM. Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus. Nat Microbiol. 2020;5(9):1088–95. pmid:32483230
- 41. Chai K, Wang Z, Pan Q, Tan J, Qiao W, Liang C. Effect of Different Nuclear Localization Signals on the Subcellular Localization and Anti-HIV-1 Function of the MxB Protein. Front Microbiol. 2021;12:675201. pmid:34093497
- 42. Cazalla D, Zhu J, Manche L, Huber E, Krainer AR, Caceres JF. Nuclear export and retention signals in the RS domain of SR proteins. Mol Cell Biol. 2002;22(19):6871–82. pmid:12215544
- 43. Greig JA, Nguyen TA, Lee M, Holehouse AS, Posey AE, Pappu RV, et al. Arginine-Enriched Mixed-Charge Domains Provide Cohesion for Nuclear Speckle Condensation. Mol Cell. 2020;77(6):1237–50 e4. pmid:32048997
- 44. Hori T, Takeuchi H, Saito H, Sakuma R, Inagaki Y, Yamaoka S. A carboxy-terminally truncated human CPSF6 lacking residues encoded by exon 6 inhibits HIV-1 cDNA synthesis and promotes capsid disassembly. J Virol. 2013;87(13):7726–36. pmid:23658440
- 45. Tian B, Manley JL. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol. 2017;18(1):18–30. pmid:27677860
- 46. Mattaj IW, Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265–306. pmid:9759490
- 47. Kosugi S, Hasebe M, Matsumura N, Takashima H, Miyamoto-Sato E, Tomita M, et al. Six classes of nuclear localization signals specific to different binding grooves of importin alpha. J Biol Chem. 2009;284(1):478–85. pmid:19001369
- 48. Cherezova L, Burnside KL, Rose TM. Conservation of complex nuclear localization signals utilizing classical and non-classical nuclear import pathways in LANA homologs of KSHV and RFHV. PLoS One. 2011;6(4):e18920. pmid:21559489
- 49. Wang L, Li M, Cai M, Xing J, Wang S, Zheng C. A PY-nuclear localization signal is required for nuclear accumulation of HCMV UL79 protein. Med Microbiol Immunol. 2012;201(3):381–7. pmid:22628116
- 50. Lange A, Mills RE, Devine SE, Corbett AH. A PY-NLS nuclear targeting signal is required for nuclear localization and function of the Saccharomyces cerevisiae mRNA-binding protein Hrp1. J Biol Chem. 2008;283(19):12926–34. pmid:18343812
- 51. Michael WM, Eder PS, Dreyfuss G. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J. 1997;16(12):3587–98. pmid:9218800
- 52. Siomi H, Shida H, Nam SH, Nosaka T, Maki M, Hatanaka M. Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell. 1988;55(2):197–209. pmid:3048703
- 53. Melen K, Keskinen P, Ronni T, Sareneva T, Lounatmaa K, Julkunen I. Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope. J Biol Chem. 1996;271(38):23478–86. pmid:8798556
- 54. Sowd GA, Serrao E, Wang H, Wang W, Fadel HJ, Poeschla EM, et al. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proc Natl Acad Sci U S A. 2016;113(8):E1054–63. pmid:26858452
- 55. Lu J, Wu T, Zhang B, Liu S, Song W, Qiao J, et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Commun Signal. 2021;19(1):60. pmid:34022911
- 56. Franke EK, Luban J. Inhibition of HIV-1 replication by cyclosporine A or related compounds correlates with the ability to disrupt the Gag-cyclophilin A interaction. Virology. 1996;222(1):279–82. pmid:8806510
- 57. Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP. Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell. 1993;73(6):1067–78. pmid:8513493
- 58. Franke EK, Yuan HE, Luban J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature. 1994;372(6504):359–62. pmid:7969494
- 59. De Iaco A, Luban J. Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA. Retrovirology. 2014;11:11. pmid:24479545
- 60. Li L, Olvera JM, Yoder KE, Mitchell RS, Butler SL, Lieber M, et al. Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J. 2001;20(12):3272–81. pmid:11406603
- 61. Chin CR, Perreira JM, Savidis G, Portmann JM, Aker AM, Feeley EM, et al. Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration. Cell Rep. 2015;13(8):1717–31. pmid:26586435
- 62. Burdick RC, Li C, Munshi M, Rawson JMO, Nagashima K, Hu WS, et al. HIV-1 uncoats in the nucleus near sites of integration. Proc Natl Acad Sci U S A. 2020;117(10):5486–5493. pmid:32094182
- 63. Luchsinger C, Lee K, Mardones GA, KewalRamani VN, Diaz-Griffero F. Formation of nuclear CPSF6/CPSF5 biomolecular condensates upon HIV-1 entry into the nucleus is important for productive infection. Sci Rep. 2023;13(1):10974. pmid:37414787
- 64. Rensen E, Mueller F, Scoca V, Parmar JJ, Souque P, Zimmer C, et al. Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages. EMBO J. 2021;40(1):e105247. pmid:33270250
- 65. Scoca V, Morin R, Collard M, Tinevez JY, Di Nunzio F. HIV-induced membraneless organelles orchestrate post-nuclear entry steps. J Mol Cell Biol. 2023;14(11). pmid:36314049
- 66. Liszewski MK, Yu JJ, O’Doherty U. Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods. 2009;47(4):254–60. pmid:19195495
- 67. Mbisa JL, Delviks-Frankenberry KA, Thomas JA, Gorelick RJ, Pathak VK. Real-time PCR analysis of HIV-1 replication post-entry events. Methods Mol Biol. 2009;485:55–72. pmid:19020818
- 68. Battivelli E, Dahabieh MS, Abdel-Mohsen M, Svensson JP, Tojal Da Silva I, Cohn LB, et al. Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4(+) T cells. Elife. 2018;7:e34655. pmid:29714165
- 69. Serrao E, Cherepanov P, Engelman AN. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites. J Vis Exp. 2016(109). pmid:27023428
- 70. Anderson-Daniels J, Singh PK, Sowd GA, Li W, Engelman AN, Aiken C. Dominant Negative MA-CA Fusion Protein Is Incorporated into HIV-1 Cores and Inhibits Nuclear Entry of Viral Preintegration Complexes. J Virol. 2019;93(21):e01118–19. pmid:31413124
- 71. Singh PK, Bedwell GJ, Engelman AN. Spatial and Genomic Correlates of HIV-1 Integration Site Targeting. Cells. 2022;11(4):655. pmid:35203306
- 72. Bedwell GJ, Jang S, Li W, Singh PK, Engelman AN. rigrag: high-resolution mapping of genic targeting preferences during HIV-1 integration in vitro and in vivo. Nucleic Acids Res. 2021;49(13):7330–46.
- 73. Carteau S, Hoffmann C, Bushman F. Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target. J Virol. 1998;72(5):4005–14. pmid:9557688
- 74. Winans S, Yu HJ, de Los Santos K, Wang GZ, KewalRamani VN, Goff SP. A point mutation in HIV-1 integrase redirects proviral integration into centromeric repeats. Nat Commun. 2022;13(1):1474. pmid:35304442
- 75. Stevens SW, Griffith JD. Human immunodeficiency virus type 1 may preferentially integrate into chromatin occupied by L1Hs repetitive elements. Proc Natl Acad Sci U S A. 1994;91(12):5557–61. pmid:8202527
- 76. Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002;110(4):521–9. pmid:12202041
- 77. Gupta K, Brady T, Dyer BM, Malani N, Hwang Y, Male F, et al. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J Biol Chem. 2014;289(30):20477–88. pmid:24904063
- 78. Marquis KA, Everett J, Cantu A, McFarland A, Sherrill-Mix S, Krystal M, et al. The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration. AIDS Res Hum Retroviruses. 2024;40(2):114–26. pmid:37125442
- 79. Kawachi T, Masuda A, Yamashita Y, Takeda JI, Ohkawara B, Ito M, et al. Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors. EMBO J. 2021;40(22):e107485. pmid:34605568
- 80. Jang S, Bedwell GJ, Singh SP, Yu HJ, Arnarson B, Singh PK, et al. HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration. Nucleic Acids Res. 2024;52(18):11060–82. pmid:39258548
- 81. Munk C, Brandt SM, Lucero G, Landau NR. A dominant block to HIV-1 replication at reverse transcription in simian cells. Proc Natl Acad Sci U S A. 2002;99(21):13843–8. pmid:12368468
- 82. Vermeire J, Naessens E, Vanderstraeten H, Landi A, Iannucci V, Van Nuffel A, et al. Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors. PLoS One. 2012;7(12):e50859. pmid:23227216
- 83. Bulli L, Apolonia L, Kutzner J, Pollpeter D, Goujon C, Herold N, et al. Complex Interplay between HIV-1 Capsid and MX2-Independent Alpha Interferon-Induced Antiviral Factors. J Virol. 2016;90(16):7469–80. pmid:27279606
- 84. Butler SL, Hansen MS, Bushman FD. A quantitative assay for HIV DNA integration in vivo. Nat Med. 2001;7(5):631–4. pmid:11329067
- 85. Brussel A, Delelis O, Sonigo P. Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA. Methods Mol Biol. 2005;304:139–54. pmid:16061972
- 86. Lawrence M, Huber W, Pages H, Aboyoun P, Carlson M, Gentleman R, et al. Software for computing and annotating genomic ranges. PLoS Comput Biol. 2013;9(8):e1003118. pmid:23950696