About

Alan N. Engelman, PhD, is a Professor of Medicine at Harvard Medical School and a member of the Engelman Lab at the Dana-Farber Cancer Institute. The provided page text does not include specific details about his research focus, background, or key contributions.

Research topics

• Computational biology
• Biology
• Virology
• Genetics
• Chemistry

Selected publications

• Development and validation of HIV SMRTcap for the characterization of HIV-1 reservoirs across tissues and subtypes

  PLoS Pathogens · 2026-01-13

  articleOpen access

  Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the global HIV/AIDS epidemic and the establishment of an integrated HIV-1 reservoir remains the primary obstacle to cure. Upon therapy interruption, reactivation of the persistent HIV-1 reservoir propagates viral rebound and mediates continued immunological decline. While furthering understanding of the HIV-1 reservoir is essential for HIV-1 cure, commonly used sequencing strategies are often limited by the reliance on short-read sequencing across separate assays to determine integration sites and proviral integrity - something that does not always adequately resolve complex human genomic repeats or low complexity regions. Simultaneous identification of proviral integration sites and proviral integrity at the single molecule level would enable HIV-1 reservoir characterization with minimal imputation or bioinformatic reconstruction. Here we present HIV Single Molecule Real Time Capture (HIV SMRTcap), a novel molecular and computational pipeline that directly and simultaneously identifies HIV-1 integration sites, defines proviral integrity, and characterizes clonal expansion of HIV-1 provirus-containing cells with single molecule resolution. In combination with long-read, single-molecule, real-time (SMRT) sequencing and custom analytic pipelines, HIV SMRTcap enables a highly comprehensive characterization of HIV-1 reservoirs. Moreover, we demonstrate here that HIV SMRTcap performs robustly across the major global subtypes (HIV-1 subtype A, B, C, D and A/D recombinant viruses), and can use both cell- and tissue-derived inputs, including samples from antiretroviral therapy (ART) treated individuals with undetectable viral loads. Our results demonstrate that HIV SMRTcap serves as a comprehensive, robust method for unbiased HIV-1 reservoir characterization. Used alone, or in combination with single-cell based methods, HIV SMRTcap will enable novel exploration of viral reservoirs across subtypes and in tissue-specific compartments, providing critical information needed to inform HIV-1 cure.

  PublisherDOI

• Integrase anchors viral RNA to the HIV-1 capsid interior

  Nature · 2026-02-18

  articleOpen access

  . Here we determined the cryogenic electron microscopy (cryo-EM) structure of a primate lentiviral IN in a complex with RNA, revealing a linear filament made of IN octamer repeat units, each comprising a pair of asymmetric homotetramers. The assembly is stabilized through IN-RNA interactions involving mainly the IN C-terminal domains and RNA backbone. The spacing and orientation of the IN filament repeat units closely matched those of consecutive capsid (CA) hexamers within the mature CA lattice. Using cryo-EM images of native purified HIV-1 cores, we refined the structure of the IN filament as it propagates along the luminal side of the CA lattice. Each IN tetramer within the filament nestled in a CA hexamer, engaging closely with the major homology regions. Substitutions of residues involved in IN-CA contacts yielded eccentric virions with RNA nucleoids located outside of the cores. Collectively, our results establish the structural basis for the HIV-1 IN-RNA interaction and reveal that IN forms an RNA-binding module on the luminal side of the mature CA lattice.

  PublisherDOI

• Relationship between the distribution of LEDGF along genes and positions of HIV-1 DNA integration

  mBio · 2026-03-04

  articleOpen access

  HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase (IN) with LEDGF, a host factor. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this activity appears inconsistent with LEDGF-mediated integration across genes and with data indicating that LEDGF promotes transcriptional elongation. We found LEDGF was enriched in peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. LEDGF harbors two globular domains, a Pro-Trp-Trp-Pro (PWWP) chromatin reader with specificity for H3K36me3, and an IN binding domain (IBD) that mediates interactions with IN and numerous cellular factors including MLL1. The IBD and MLL1 mediated LEDGF recruitment to promoters. In turn, LEDGF promoted the association of RNA Pol II at TSSs. Consistent with greater enrichment of H3K36me3 at the 3' regions of genes, LEDGF lacking the PWWP domain had reduced association with downstream sequences and increased enrichment at TSSs. HIV-1 integration levels per gene revealed that a threshold amount of LEDGF at TSSs was associated with integration in downstream sequence. It is thought that the PWWP domain and the enrichment of H3K36me3 in transcribed sequences are responsible for integration across genes. Although the distribution of HIV-1 integrations across gene bodies was shifted upstream in cells lacking H3K36me3, integration levels per gene were unchanged. Our results support a model where LEDGF is tethered to promoters via IBD-mediated cell factor interactions. Subsequently, LEDGF associates with RNA Pol II during elongation to effect HIV-1 integration site targeting.IMPORTANCEOver 40 million people are currently infected with HIV-1, and approximately one million new infections occur each year. While antiretroviral drugs are extremely successful in suppressing HIV-1, drug resistance is increasing, and there are no reasonable approaches to cure patients of the virus. The chromatin-associated transcription factor LEDGF interacts directly with viral integrase (IN), causing HIV-1 integration to occur across the bodies of actively transcribed genes. The research here identifies the molecular determinants that position LEDGF at promoters and across genes. Our experiments discovered that LEDGF at promoters does not mediate integration. These results may lead to the identification of factors and interactions that inhibit integration and offer the potential to develop antiviral therapies that suppress HIV-1 replication.*Corresponding author.

  PublisherDOI

• Retroviral intasome architecture shapes the dynamics of target DNA search and integration

  PLoS Pathogens · 2026-03-20

  articleOpen access

  Recombinant retroviral intasomes assembled from purified integrase (IN) and oligonucleotides mimicking viral DNA ends (vDNA) faithfully recapitulate concerted integration in vitro. Structural studies of retroviral intasomes have revealed an array of IN oligomer forms, which appear to share a conserved intasome core coordinating the vDNA ends for strand transfer into target DNA. Here we have explored the biochemical and dynamic properties of the mouse mammary tumor virus (MMTV) octameric intasome. We show that MMTV intasomes continue to accumulate concerted integration products for ~80 min in vitro, whereas prototype foamy virus (PFV) intasomes plateau within ~2 min. MMTV integration activity peaks within the range of physiological ionic strength and is more active in the presence of manganese compared to magnesium. Single-molecule images demonstrate that the target DNA search by MMTV intasomes appears rate-limiting, similar to PFV intasomes. The time between strand transfer of the two MMTV vDNA ends into the target DNA is ~ 3 fold slower than PFV intasomes. This is the first report of the dynamics of an orthoretrovirus intasome interacting with target DNA with single molecule resolution.

  PublisherDOI

• Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity

  Nucleic Acids Research · 2025-05-16 · 1 citations

  articleOpen access

  Selection of a suitable chromatin environment during retroviral integration is a tightly regulated process. Most retroviruses, including spumaretroviruses, require mitosis for nuclear entry. However, whether intrinsic chromatin dynamics during mitosis modulates retroviral genome invasion is unknown. Previous work uncovered critical interactions of prototype foamy virus (PFV) Gag with nucleosomes via a highly conserved arginine anchor residue. Yet, the regulation of Gag-chromatin interaction and its functional consequences for spumaretrovirus biology remain obscure. Here, we investigated the kinetics of chromatin binding by Gag during mitosis and proviral integration in synchronized cells. 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 chromosomes. Mutant Gag proteins were, moreover, defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed chromatin. These data indicate that the chromatin landscape during Gag-nucleosome interactions is important for PFV integration site selection and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation.

  PublisherDOI

• Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-11

  preprintOpen access

  Abstract 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.

  PublisherOA PDFDOI

• CPSF6 Promotes HIV-1 Preintegration Complex Function

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-29

  preprintOpen access

  ABSTRACT Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as a HIV-1 capsid (CA) binding host factor and promotes viral DNA integration targeting into gene dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC) - the machinery that carries out viral DNA integration to establish infection - is unknown. To study CPSF6’s role in HIV-1 PIC function, we extracted PICs from cells depleted of CPSF6 or expressing a CPSF6 mutant that cannot bind to CA. These PICs exhibited significantly lower integration activity when compared to the control PICs. Addition of recombinant CPSF6 restored the integration activity of PICs extracted from the mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6’s effect on PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in viral integration in these cells was detected and this defect was not a consequence of reduced reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6 mutant cells. Finally, sequencing analysis revealed that HIV-1 integration in the CPSF6 mutant cells was significantly redirected from the gene dense regions of the host genome. Collectively, these results suggest that CPSF6-CA interaction regulates PIC function both in vitro and in infected cells. IMPORTANCE HIV-1 infection is dependent on the interaction of the virus with host factors. However, the molecular details of virus-host factor interactions are not fully understood. For instance, HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 that restricted HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes integration targeting into gene dense regions of the host genome. Here we report that CPSF6-CA interaction promotes the activity of HIV-1 preintegration complexes (PICs). We also observed that disruption of CPSF6-CA binding in target cells significantly reduced viral integration and directed integration targeting away from gene-dense regions. These findings demonstrate a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

  PublisherOA PDFDOI

• CPSF6 promotes HIV-1 preintegration complex function

  Journal of Virology · 2025-04-09 · 3 citations

  articleOpen access

  ABSTRACT Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as an HIV-1 capsid (CA) binding host factor to promote viral DNA integration targeting into gene-dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC)—the sub-viral machinery that carries out viral DNA integration—are unknown. To study CPSF6’s role in HIV-1 PIC function, we extracted PICs from cells that are either depleted of CPSF6 or express a mutant form that cannot bind to CA. These PICs exhibited significantly lower viral DNA integration activity when compared to the control PICs. The addition of purified recombinant CPSF6 restored the integration activity of PICs extracted from the CPSF6-mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6’s role in PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in integration in these cells was detected, and this reduction was not a consequence of lower reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA–CPSF6 binding showed no integration defect in CPSF6-mutant cells. Finally, sequencing analysis revealed that HIV-1 integration into CPSF6-mutant cell genomes was significantly redirected away from gene-dense regions of chromatin compared to the control cells. Collectively, these results suggest that the CPSF6–CA interaction promotes PIC function both in vitro and in infected cells. IMPORTANCE HIV-1 infection is dependent on the interaction of the virus with cellular host factors. However, the molecular details of HIV–host factor interactions are not fully understood. For instance, the HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 to restrict HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes HIV-1 integration targeting into gene-dense regions of the host genome. Here, we provide evidence that CPSF6–CA interaction stimulates the activity of HIV-1 preintegration complexes (PICs). We also describe that disruption of CPSF6–CA binding in target cells significantly reduces viral DNA integration and redirects integration targeting away from gene-dense regions into regions of low transcriptional activity. These findings identify a critical role for the CPSF6–CA interaction in PIC function and integration targeting.

  PublisherDOI

• Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins

  mBio · 2025-01-24 · 2 citations

  articleOpen access

  ABSTRACT Interlinked interactions between the viral capsid (CA), nucleoporins (Nups), and the antiviral protein myxovirus resistance 2 (MX2/MXB) influence human immunodeficiency virus 1 (HIV-1) nuclear entry and the outcome of infection. Although RANBP2/NUP358 has been repeatedly identified as a critical player in HIV-1 nuclear import and MX2 activity, the mechanism by which RANBP2 facilitates HIV-1 infection is not well understood. To explore the interactions between MX2, the viral CA, and RANBP2, we utilized CRISPR-Cas9 to generate cell lines expressing RANBP2 from its endogenous locus but lacking the C-terminal cyclophilin (Cyp) homology domain and found that both HIV-1 and HIV-2 infections were reduced significantly in RANBP2 ΔCyp cells. Importantly, although MX2 still localized to the nuclear pore complex in RANBP2 ΔCyp cells, antiviral activity against HIV-1 was decreased. By generating cells expressing specific point mutations in the RANBP2-Cyp domain, we determined that the effect of the RANBP2-Cyp domain on MX2 anti-HIV-1 activity is due to direct interactions between RANBP2 and CA. We further determined that CypA and RANBP2-Cyp have similar effects on HIV-1 integration targeting. Finally, we found that the Nup requirements for HIV infection and MX2 activity were altered in cells lacking the RANBP2-Cyp domain. These findings demonstrate that the RANBP2-Cyp domain affects viral infection and MX2 sensitivity by altering CA-specific interactions with cellular factors that affect nuclear import and integration targeting. IMPORTANCE Human immunodeficiency virus 1 (HIV-1) entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid (CA) and multiple cellular proteins, including nucleoporins (Nups) such as RANBP2. Nups also mediate the function of the antiviral protein myxovirus resistance 2 (MX2); however, determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore complex (NPC) and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the cyclophilin domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.

  PublisherDOI

• The nuclear localization signal of CPSF6 governs post-nuclear import steps of HIV-1 infection

  PLoS Pathogens · 2025-01-17 · 7 citations

  articleOpen accessCorresponding

  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.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01AI045313

  NIH · $586k · 2003

• NIH Grant P50GM103368

  NIH · $39.8M · 2017

• Cryo-Electron Microscopy and Tomography Core

  NIH · $28.1M · 2007–2023

• NIH Grant U54GM103368

  NIH · $22.0M · 2022

• NIH Grant R21AI045313

  NIH · $66k · 2000

Frequent coauthors

• Peter Cherepanov

  99 shared

• Wen Li

  Chengdu University of Traditional Chinese Medicine

  76 shared

• Sooin Jang

  Dana-Farber Cancer Institute

  71 shared

• Parmit K. Singh

  Dana-Farber Cancer Institute

  64 shared

• Mamuka Kvaratskhelia

  University of Colorado Anschutz Medical Campus

  58 shared

• Gregory J. Bedwell

  Dana-Farber Cancer Institute

  52 shared

• Peijun Zhang

  Diamond Light Source

  52 shared

• Richard Lu

  Ragon Institute of MGH, MIT and Harvard

  49 shared

Labs

• Engelman LabPI

  Not provided

Education

• PhD, Molecular Biology and Microbiology

  Tufts University School of Medicine

  1990