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Singh, Parmit

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Singh, Parmit
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    LEDGF/P75 DIRECTS HIV-1 TO INTEGRATE INTO HIGHLY SPLICED GENES DESPITE THEIR COMPARATIVELY LOW EXPRESSION
    Singh, Parmit; Engelman, Alan
    Integration of HIV-1 into genes depends on multiple factors including transcription, splicing, LEDGF/p75, and CPSF6. But, the precise role of mRNA splicing in HIV-1 integration targeting is unclear. Transcriptional intron content can show high rate of splicing due to high transcription rate, whereas genes with higher number of introns can show high rate of splicing due to intron number, despite comparatively low transcription rate. To differentiate the roles of splicing and transcription in HIV-1 integration, we compared gene populations targeted by HIV-1 to random gene populations generated in silico (RIC, for random integration control). Analyzing gene groups with same numbers of introns, we determined that genes containing >10 introns were preferentially enriched for HIV-1 integration (ratio >1; R2 =0.9). We then parsed the human transcriptome into genes containing 0-10 introns (71% of all genes) and genes with minimally 11 introns (29% of all genes). Remarkably, we found that genes with >10 introns harbored 57% of all HIV-1 integration sites compare to 35% of sites in genes with <11 introns. This preference was independent of gene length: length-normalized genes with <11 introns were targeted 1.3 times greater than random whereas genes with >10 introns were targeted 2.1 times over the RIC (P < e-10). To assess the roles of virus-host interactions, we analyzed integration sites from LEDGF knockout (LKO) and CPSF6 knockout (CKO) cell lines. For genes with >10 introns, integration was similarly reduced by 20-21% in both cell types. Integration into genes with <11 introns was by contrast reduced by 2% and 7.6% in LKO and in CKO cells, respectively, indicating that LEDGF/p75 preferentially directs integration to genes with >10 introns. Consistent with this interpretation, analysis of a recently published LEDGF/p75 ChIP-Seq dataset revealed a strong correlation between number of genic LEDGF/p75 interaction sites and intron number (R2 =0.7; P < e-5), with 14 as the average number of introns for genes with minimally one LEDGF/p75 binding site. In LKO cells, integration into genes with LEDGF/p75 interaction sites was reduced by 14% (12% in genes with >10 introns), while integration into genes that lacked LEDGF/p75 interaction sites was reduced by 6% and 21% in LKO and CKO cells, respectively. Moreover, integration into genes with <11 introns that lacked LEDGF/p75 interaction sites was reduced in CKO but not in LKO cells. Our results reveal a previously unrecognized role for LEDGF/p75 in mediating HIV-1 integration into highly spliced genes that are expressed at comparatively low levels.
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    mRNA splicing targets HIV integration into PAF-1-regulated genes.
    (2024-03-01) Singh, Parmit; Annamalai, Arun; Kvaratkshelia, Mamuka; Engelman, Alan
    Pre-mRNA co-transcriptional splicing is coupled with promoter-proximal Pol II pausing and alternative polyadenylation (APA)1,2. Splicing inhibitors increase pausing and the use of upstream or proximal polyadenylation sites (PASs) by impairing the recruitment of positive transcription elongation factor b (P-TEFb)1,2, which is a core component of the super elongation complex (SEC). The cleavage factor Im (CFIm) complex consisting of cleavage and polyadenylation specificity factor (CPSF) 6 and CPSF5 regulates APA by promoting the use of distal PASs. CPSF6 binds capsid (CA) to license HIV-1 intranuclear trafficking and integration targeting into highly spliced or intron rich genes. Based on the interconnections between splicing, pausing, and APA, we hypothesized that APA and Pol II elongation might play a role in HIV-1 integration targeting. Indeed, in Jurat T cells3, APA-regulated genes dependent on U2 snRNP (4.6% of human genes) for the selection of distal PASs harbored 24% of HIV-1 integration sites (3x compared to RIC or random integration control; p<1E-5). In contrast, genes independent of U2 snRNP for APA regulation were targeted similarly to all human genes (p< 0.2). Apart from splicing and CFIm complex, Pol II associated factor-1 (PAF-1) regulates pausing4 and the selection of distal PASs5. Additionally, PAF-1 regulates the expression of integrated proviral DNA. We observed that paused genes regulated by PAF-1 (14% of human genes) were preferentially targeted (3.5x RIC, containing 40% HIV-1 sites; p<1E-5), whereas the reciprocal gene set was preferentially avoided (p<1E-5). To test the role of splicing, we infected Jurkat T cells in the presence of the U2 snRNP inhibitor Pladienolide B (Plad B) or the SEC inhibitor KL-2 and determined sites of HIV-1 integration. Whereas Plad B significantly reduced integration into PAF-1 paused genes, it failed to significantly effect integration into the reciprocal set of human genes. We called chromosomes with reduced genic integrations (p<1E-04) as Plad B sensitive chromosomes (PBSC) and the remaining chromosomes with increased genic integrations as Plad B insensitive chromosomes (PBIC; p<0.02)). Although both PBSC and PBIC genes had the same average number of introns, PBSC were comparatively gene enriched (12 genes/Mb) whereas PBIC were gene-poor (7 genes/Mb; the average genes/Mb in human genome is 9). KL-2 reduced genic integration significantly for PBSC but not for PBIC, suggesting that both splicing and SEC inhibitors reduced HIV-1 integration into the same sets of genes. To test the roles of integration targeting cofactors, we mapped sites for CPSF6-defective CA mutant viruses or wild type (WT) HIV-1 in LEDGF/p75 knockout (LKO) cells. PBSC supported significantly less genic integration for CA mutants and for WT virus in LKO cells (p<1E-7). However, while PBIC were significantly less targeted by WT virus in LKO cells, these genes were significantly more targeted by CA mutants (p<1E-7 for both comparisons). Thus, the CPSF6-CA interaction is critical for preferential HIV-1 integration targeting of paused genes and APA genes regulated through P-TEFb/SEC and splicing. Currently, we are planning to differentiate the role of CPSF6-dependent APA from CPSF6-dependent trafficking of CA in the targeting of paused and APA-regulated genes.
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    HIV-1 Preferentially Targets Genes Regulated by PAF-1 and U2 snRNP for Integration
    (2023-08-04) Singh, Parmit; Annamalai, Arun; Kvaratkshelia, Mamuka; Engelman, Alan
    Pre-mRNA splicing is coupled with promoter-proximal Pol II pausing and alternative polyadenylation (APA). Splicing inhibitors increase pausing and the use of proximal polyadenylation sites (PAS) in intron rich genes by impairing P- TEFb recruitment, which is a core component of the super elongation complex (SEC). The CFIm complex consisting of CPSF6 and CPSF5 also regulates APA by promoting the use of distal PAS. CPSF6 binds viral capsid (CA) to license HIV- 1 intranuclear trafficking and integration targeting. Previously, we showed that HIV-1 preferentially integrates into intron rich, Pol II-paused genes. Based on the interconnections between splicing, pausing, and APA, we hypothesized that APA might play a role in HIV-1 integration targeting. Indeed, in Jurat T cells, APA genes regulated by U2 snRNP contained 24% of integration sites (3x compared to RIC or random integration control; p<1E-5). In contrast, nonregulated genes were targeted similarly to all genes (p< 0.2). Further, paused genes regulated by PAF- 1, which is also important for APA and for post-integration viral expression, were preferentially targeted (3.5x RIC; p<1E-5), whereas the reciprocal gene set was preferentially avoided (p<1E-5). To test the role of splicing, we infected Jurkat T cells in the presence of the U2 snRNP inhibitor Pladenolide B (Plad B) or the SEC inhibitor KL-2. Plad B significantly reduced genic integration in PAF-1 paused genes but not in unpaused genes. We defined chromosomes with reduced genic integrations (p<1E-04) as Plad B sensitive chromosomes (PBSC) and the remaining chromosomes as Plad B insensitive chromosomes (PBIC; p<0.02)). KL-2 reduced genic integration significantly for PBSC but not for PBIC, suggesting that splicing targets HIV-1 integration into genes regulated by P-TEFb/SEC. To test the roles of integration targeting cofactors, we mapped sites for CPSF6- defective CA mutant viruses or wild type (WT) HIV-1 in LEDGF/p75 knockout (LKO) cells. PBSC supported significantly less genic integration for CA mutants and for WT virus in LKO cells (p<1E-7). However, while PBIC were significantly less targeted by WT virus in LKO cells, these genes were significantly more targeted by CA mutants (p<1E-7 for both comparisons). Thus, the CPSF6-CA interaction is critical for preferential HIV-1 integration targeting of paused genes and APA genes regulated through P-TEFb/SEC.
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    CPSF6 LICENSES HIV-1 INTEGRATION INTO POL II-PAUSED GENES REGULATED by P-TEFb and U2 snRNP
    (2023-05) Singh, Parmit; Annamalai, Arun; Kvaratskhelia, Mamuka; Engelman, Alan
    Pre-mRNA splicing regulates promoter-proximal Pol II pausing and alternative polyadenylation (APA). Splicing inhibitors increase pausing and use of proximal polyadenylation sites (PAS) for comparatively long (>4 introns) transcripts by impairing P-TEFb recruitment, which is a core component of the super elongation complex (SEC). Similar to splicing, the CFIm complex composed of CPSF6 and CPSF5 promotes the use of distal PAS. CPSF6 binds capsid (CA) to target integration into highly spliced genes and gene-dense (GD) regions. We previously reported that HIV-1 preferentially integrates into genes harboring >10 introns and Pol II-paused genes. Based on the connection between intron number, pausing, and APA, we hypothesized that CPSF6 targets HIV-1 integration into paused genes regulated by U2 snRNP and P-TEFb. U2 snRNP-regulated APA genes, which represent 5% of the transcribed genome, contained 24% of integration sites (3x compared to RIC or random integration control; p<1E-5). In contrast, nonregulated genes were targeted similarly to all genes (p< 0.2). Moreover, we found that paused genes regulated by PAF-1, which is important for APA, were preferentially targeted (3.5x RIC; p<1E-5), whereas the reciprocal gene set was preferentially avoided (p<1E-5). To test the role of splicing, we mapped integration sites in Jurkat T cells in the presence of the U2 snRNP inhibitor Pladenolide B (Plad B) or SEC inhibitor KL-2. Plad B significantly reduced genic integration in PAF-1 paused genes but not in unpaused genes. We defined chromosomes with reduced genic integrations as Plad B sensitive chromosomes (PBSC) and the remaining chromosomes as Plad B insensitive chromosomes (PBIC). KL-2 reduced genic integration significantly for PBSC but not for PBIC, suggesting that splicing targets HIV-1 into genes regulated by P-TEFb. To test the roles of integration targeting cofactors, we mapped sites for CPSF6- defective CA mutant viruses or WT HIV-1 in LEDGF/p75 knockout (LKO) cells. Despite predictably similar levels of splicing, PBSC (avg. intron content 9.6 and GD 11.8/Mb) were targeted 2.6x to RIC, whereas PBIC (avg. intron 9.5 and 6.6 genes/Mb) were but 1.2x. PBSC supported significantly less genic integration for CA mutants and for WT virus in LKO cells (p<1E-7). However, while PBIC were significantly less targeted by WT virus in LKO cells, these genes were significantly more targeted by CA mutants (p<1E-7 for both comparisons). Thus, CPSF6-CA interactions preferentially target HIV-1 integration into paused genes regulated by the SEC and U2 snRNP. Acknowledgment: HUCFAR 5P30AI060354-18.
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    HIV-1 Integration Favors Genes Associated With Promoter-Proximal Pol II Pausing
    (2021-05) Singh, Parmit; Annamalai, Arun; Kvaratkshelia, Mamuka; Engelman, Alan
    HIV-1 integration targeting is mediated by interactions of viral integrase and capsid with LEDGF/p75 and CPSF6, respectively. HIV-1 also prefers to integrate into highly spliced genes, but the role of pre-mRNA splicing in HIV-1 integration is unclear. Splicing inhibitors reduce the association of exon junction complex (EJC) with genes containing introns but not with intronless genes. The EJC causes promoter-proximal Pol II pausing, which is released by super elongation complex (SEC). To assess the role of Pol II pausing in integration, genes were stratified as paused versus unpaused, which revealed 50%/40% respective HIV-1 integration targeting preferences in HEK293T cells; random paused and unpaused values were 15%/34%. In LEDGF/p75 knockout (LKO), CPSF6 knockout (CKO), and double knockout (DKO) cells, unpaused gene targeting dropped marginally from 40% to 37%, 38%, and 35%, respectively. However, in paused genes, 50% targeting reduced to 31%, 24%, and 20% in respective LKO, CKO and DKO cells. Thus, HIV-1 prefers integration into Pol II-paused genes. To test the role of splicing, we mapped integration sites in the presence of splicing inhibitor Pladienolide B (PladB) in WT and LKO Jurkat T cells. Genic integration in WT cells was reduced in a dose-dependent manner, with maximum reduction of 2.7% (P <2E-31) at 9 nM. In LKO cells, genic integration was reduced by 3.6% (P <4E-13), but only at 9 nM inhibitor, suggesting both LEDGF/p75-dependent and independent roles for pre- mRNA splicing in integration targeting. Similar to HEK293T cells, integration in paused and unpaused genes was 55% and 38% in WT Jurkat cells, while in LKO cells, these respective values were 25% and 35%. PladB significantly reduced integration into paused genes in both WT and LKO, but only at 9 nM in LKO cells. By contrast, whereas PladB significantly increased integration into unpaused genes (1.2% at 9 nM; P <2E-16) in WT cells, it had no effect in LKO cells. To rule out the role of pausing itself, we assessed the SEC inhibitor KL-2 in HEK293T cells, which revealed significant reductions in the amount of integration in genes, paused genes, and speckle-associated domains. Yet, integration into unpaused genes was unaffected. Thus, our results reveal an interplay between pre-mRNA splicing with Pol II pausing and transcriptional elongation in HIV-1 integration targeting. Acknowledgment: Harvard University Center for AIDS Research (HU CFAR NIH/NIAID fund 5P30AI060354-17).
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    LEDGF/p75-dependent HIV-1 integration targeting into spliced genes: A role for the DNA damage response
    (2020-04) Singh, Parmit; Engelman, Alan
    HIV-1 integration site selection is regulated at the level of transcription, splicing, histone modifications, super enhancer, nuclear architecture, and interactions of viral proteins with host factors. However, the mechanism of integration is not fully understood. Further, we lack a common understanding how these factors work together. HIV-1 integrase interacts with LEDGF/p75 and viral capsid interacts with another host factor CPSF6. Both interactions are important for HIV-1 integration in active genes, spliced genes and in gene-dense regions. LEDGF/p75 also interacts with several splicing factors (SFs) but we have not yet identified any SF with its possible role in HIV-1 integration. Therefore, we don’t understand how and why splicing plays a role in HIV-1 integration site selection. Here, we show that genes > 10 introns are highly targeted for integration. Further, we identified 15 different subsets of spliced genes that are less targeted in the absence of LEDGF/p75 or CPSF6 compared to WT cells. SFs that regulate these 15 spliced gene subsets are enriched for DNA damage response (DDR) proteins, suggesting a potential role of splicing factors in the repair of viral 5’ ends during integration. Moreover, we have determined that 160 out of 342 (46.8%) LEDGF/p75-interacting SFs1 are involved in DDR, suggesting enrichment of DDR proteins in LEDGF/p75-interacting SFs (P-value 4E-211, hypergeometric distribution, 367 DDR genes in population of 25,000 human genes). Additionally, we showed that alternative polyadenylation (APA) also plays a role in HIV-1 integration. Thus our results suggest that APA/splicing linked with DDR plays a role in HIV-1 integration. Recent study2 showed that genes with more than 1 integration in at least 2 out of 8 datasets are proximal to a super enhancer (SE). SEs contribute to 3D genome organization that is a driving force of integration in highly targeted genes. Our studies showed that all RefSeq genes of human hg19 were uniformly distanced from SEs. Genes > 10 introns, genes < 11 introns, intronless genes1 (least targeted) and highly targeted cancer genes1 were at the similar distance from SE, suggesting that highly targeted and less targeted genes are at similar distances from SEs. To test the role of SFs in integration targeting and 5’ end gap repair, we are knocking out/knocking down selective SFs to quantify3 associated defects.
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    Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity
    (Cold Spring Harbor Laboratory, 2025-01-11) Singh, Parmit; Lagadec, F.; Calmels, C.; Lapaillerie, D.; Lindermann, D.; Parissi, V.; Cherepanov, P.; Engelman, A.N.; Lesbats, P.
    Selection of a suitable chromatin environment during retroviral integration is a tightly regulated and multilayered process that involves interplay between viral and host factors. However, whether intrinsic chromatin dynamics during mitosis modulate retroviral genome invasion is currently poorly described. Direct interaction between the spumaretrovirus prototype foamy virus (PFV) Gag protein and cellular chromatin has been described as a major determinant for integration site selection. A previous Gag chromatin-binding site (CBS)–nucleosome co-crystal structure revealed an interaction with the histone H2A-H2B acidic patch via a highly conserved arginine anchor residue. Yet, the molecular mechanisms regulating Gag-chromatin capture during PFV infection remain obscure. Here, we investigated the kinetics of Gag-chromatin interactions during mitosis and proviral integration of PFV-infected synchronized cells. Using Gag CBS variant viruses, we showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity and redistributed viral integration sites to markers associated with late replication timing of host chromosomes. Mutant Gag proteins were moreover defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed mitotic chromatin. These data indicate that the mitotic chromatin landscape during Gag–nucleosome interactions hosts PFV integration site selection determinants and that spumaretroviruses evolved high- affinity chromatin binding to overcome early mitosis chromatin condensation for optimal viral DNA tethering, integration and infection.
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    Spatial and Genomic Correlates of HIV-1 Integration Site Targeting
    (MDPI AG, 2022-02-14) Singh, Parmit; Bedwell, Gregory; Engelman, Alan
    HIV-1 integrase and capsid proteins interact with host proteins to direct preintegration complexes to active transcription units within gene-dense regions of chromosomes for viral DNA integration. Analyses of spatially-derived genomic DNA coordinates, such as nuclear speckle-associated domains, lamina-associated domains, super enhancers, and Spatial Position Inference of the Nuclear (SPIN) genome states, have further informed the mechanisms of HIV-1 integration targeting. Critically, however, these different types of genomic coordinates have not been systematically analyzed to synthesize a concise description of the regions of chromatin that HIV-1 prefers for integration. To address this informational gap, we have extensively correlated genomic DNA coordinates of HIV-1 integration targeting preferences. We demonstrate that nuclear speckle-associated and speckle-proximal chromatin are highly predictive markers of integration and that these regions account for known HIV biases for gene-dense regions, highly transcribed genes, as well as the mid-regions of gene bodies. In contrast to a prior report that intronless genes were poorly targeted for integration, we find that intronless genes in proximity to nuclear speckles are more highly targeted than are spatially-matched intron-containing genes. Our results additionally highlight the contributions of capsid and integrase interactions with respective CPSF6 and LEDGF/p75 host factors in these HIV-1 integration targeting preferences.
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    Pre-mRNA Splicing and Gene Density are Significant Determinants of Lentiviral Integration
    (2021-05) Singh, Parmit; Chivukula, Vidya; Cherepanov, Peter; Engelman, Alan
    Nuclear speckles harbor proteins important for transcription and pre-mRNA splicing. HIV-1 preintegration complexes localize to speckles in a capsid (CA)-CPSF6 dependent manner and preferentially target speckle-associated domains (SPADs) for integration. Because the CPSF6-CA interaction is also crucial for integration into gene-dense regions and spliced genes, we correlated the extents of SPAD targeting with gene-dense regions and spliced genes. Our bioinformatic analysis mapped SPADs to gene-dense regions. While the average gene density of the human genome is 9 genes/Mb, nearly 98% of SPADs mapped to regions with gene density > 9 genes/Mb. Our analysis revealed that human chr17 and 19 contained the highest number of genes, intron-containing genes, SPADs, pol II sites, and H3K36me3 sites per Mb. Analysis of HIV-1 integration sites revealed enrichment on chr17 and 19 in wildtype but not in CPSF6 knockout cells. Sites of LEDGF/p75 occupancy and lamina-associated domains were not enriched on human chr17 and 19. Non-primate lentiviruses EIAV, FIV, BIV only marginally targeted SPADs for integration in human cells. As expected, these viruses did not preferentially target gene-dense human chr17 and 19. We next asked whether these lentiviruses targeted gene-dense regions in their natural hosts. As control, the primate lentivirus SIV preferentially targeted gene-dense chr17 and 19 in human cells and gene-dense chr16 and 19 in rhesus cells. Further, the ovine lentivirus MVV did not preferentially target human chr17 and 19 but targeted gene-dense chr11 in the sheep genome. Plausibly, species-specific host factors help guide primate versus non-primate lentiviruses to target gene-dense chromosomal regions for integration. We are currently determining non-primate lentiviral integration sites in additional native host cells. We next asked whether MVV targets spliced genes in sheep. We determined a high correlation (R2=0.6) between integration per kb and the number of sheep introns. By contrast, this correlation was low (R2=0.2) for MVV in human cells due to the lack of SPAD targeting. SPAD-associated human genes contained 0.4 introns per kb, whereas SPAD non-associated genes contained 0.1 introns per kb (P<2E-16), suggesting that SPAD- associated genes are highly spliced. These data suggest a conserved role for pre-mRNA splicing in both primate and non-primate lentiviral replication and indicates that the viruses utilize species-specific factors for trafficking to their preferred integration sites. Acknowledgment: Harvard University Center for AIDS Research (HU CFAR NIH/NIAID fund 5P30AI060354-17).
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    Allosteric Integrase Inhibitor Influences on HIV-1 Integration and Roles of LEDGF/p75 and HDGFL2 Host Factors
    (MDPI, 2022-08) Singh, Parmit; Li, Wen; Bedwell, Gregory J.; Fadel, Hind J.; Poeschla, Eric M.; Engelman, Alan N.
    Allosteric integrase (IN) inhibitors (ALLINIs), which are promising preclinical compounds that engage the lens epithelium-derived growth factor (LEDGF)/p75 binding site on IN, can inhibit different aspects of human immunodeficiency virus 1 (HIV-1) replication. During the late phase of replication, ALLINIs induce aberrant IN hyper-multimerization, the consequences of which disrupt IN binding to genomic RNA and virus particle morphogenesis. During the early phase of infection, ALLINIs can suppress HIV-1 integration into host genes, which is also observed in LEDGF/p75- depelted cells. Despite this similarity, the roles of LEDGF/p75 and its paralog hepatoma-derived growth factor like 2 (HDGFL2) in ALLINI-mediated integration retargeting are untested. Herein, we mapped integration sites in cells knocked out for LEDGF/p75, HDGFL2, or both factors, which revealed that these two proteins in large part account for ALLINI-mediated integration retargeting during the early phase of infection. We also determined that ALLINI-treated viruses are defective during the subsequent round of infection for integration into genes associated with speckle-associated domains, which are naturally highly targeted for HIV-1 integration. Class II IN mutant viruses with alterations distal from the LEDGF/p75 binding site moreover shared this integration retargeting phenotype. Altogether, our findings help to inform the molecular bases and consequences of ALLINI action.