Person:
Engelman, Alan

Profile Picture

Email Address

AA Acceptance Date

Birth Date

Research Projects

Organizational Units

Job Title

Last Name

Engelman

First Name

Alan

Name

Engelman, Alan
Author's Publications: search.filters.isAuthorOfPublication.3b734007-4ba8-48f6-acab-68c12fdcbc92

Search Results

Now showing 1 - 10 of 27
  • No Thumbnail Available
    Publication
    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.
  • No Thumbnail Available
    Publication
    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.
  • No Thumbnail Available
    Publication
    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.
  • No Thumbnail Available
    Publication
    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.
  • No Thumbnail Available
    Publication
    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).
  • No Thumbnail Available
    Publication
    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.
  • Thumbnail Image
    Publication
    CryoEM structure of MxB reveals a novel oligomerization interface critical for HIV restriction
    (American Association for the Advancement of Science, 2017) Alvarez, Frances J. D.; He, Shaoda; Perilla, Juan R.; Jang, Sooin; Schulten, Klaus; Engelman, Alan; Scheres, Sjors H. W.; Zhang, Peijun
    Human dynamin–like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein–fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction.
  • Thumbnail Image
    Publication
    Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding
    (Oxford University Press, 2014) Serrao, Erik; Krishnan, Lavanya; Shun, Ming-Chieh; Li, Xiang; Cherepanov, Peter; Engelman, Alan; Maertens, Goedele N.
    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration.
  • Thumbnail Image
    Publication
    Biochemical Characterization of Novel Retroviral Integrase Proteins
    (Public Library of Science, 2013) Ballandras-Colas, Allison; Naraharisetty, Hema; Li, Xiang; Serrao, Erik; Engelman, Alan
    Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.
  • Thumbnail Image
    Publication
    Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity
    (Public Library of Science, 2013) Matreyek, Kenneth A.; Yücel, Sara S.; Li, Xiang; Engelman, Alan
    Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.