HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration.

HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.

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

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.

The role of LEDGF in transcription is exploited by HIV-1 to position integration.

HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Brief Histories of Retroviral Integration Research and Associated International Conferences.

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.

Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, informing antiretroviral therapies that contain lenacapavir.

Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

Capsid-host interactions for HIV-1 ingress.

The HIV-1 capsid, composed of approximately 1,200 copies of the capsid protein, encases genomic RNA alongside viral nucleocapsid, reverse transcriptase, and integrase proteins. After cell entry, the capsid interacts with a myriad of host factors to traverse the cell cytoplasm, pass through the nuclear pore complex (NPC), and then traffic to chromosomal sites for viral DNA integration. Integration may very well require the dissolution of the capsid, but where and when this uncoating event occurs remains hotly debated. Based on size constraints, a long-prevailing view was that uncoating preceded nuclear transport, but recent research has indicated that the capsid may remain largely intact during nuclear import, with perhaps some structural remodeling required for NPC traversal. Completion of reverse transcription in the nucleus may further aid capsid uncoating. One canonical type of host factor, typified by CPSF6, leverages a Phe-Gly (FG) motif to bind capsid. Recent research has shown these peptides reside amid prion-like domains (PrLDs), which are stretches of protein sequence devoid of charged residues. Intermolecular PrLD interactions along the exterior of the capsid shell impart avid host factor binding for productive HIV-1 infection. Herein we overview capsid-host interactions implicated in HIV-1 ingress and discuss important research questions moving forward. Highlighting clinical relevance, the long-acting ultrapotent inhibitor lenacapavir, which engages the same capsid binding pocket as FG host factors, was recently approved to treat people living with HIV.

Pathways and Intersections: Multifaceted Approaches to Engage Individuals From Underrepresented and Marginalized Communities in HIV Research and Career Development.

BACKGROUND: The underrepresentation of historically marginalized groups in the HIV research workforce is a barrier to reaching national Ending the Epidemic goals. SETTING: The Harvard University Center for AIDS Research (HU CFAR) Diversity Equity and Inclusion Working Group (DEI WG) uses a multifaceted approach to enhance the field's diversity. METHODS: We established a DEI WG to improve the recruitment, inclusion, and retention of underrepresented minorities (URMs) in HIV research. We use community-based, participatory processes to establish and expand education and outreach programs about HIV care and research to better connect the HU CFAR to communities affected by HIV. This article reports on the development of the WG in July 2022, progress in its first year, and future plans. RESULTS: We have built a network of >50 investigators across the university for monthly meetings; partnered with existing research pathway programs for high school, undergraduate, and graduate students, directly supporting 7 new trainees and linking CFAR investigators to additional mentorship opportunities; and created 2-year Scholar Awards for 5 URM investigators in HIV. Planned work includes needs assessments for early-stage investigators to understand factors contributing to inclusion and retention and new pathway and outreach programming being developed with community partner minority-serving institutions. CONCLUSIONS: The HU CFAR DEI WG strives to ensure that individuals from underrepresented, marginalized, and minoritized communities have an opportunity to contribute to HIV research and that research is informed by the needs of the communities affected by the epidemic. An intersectional approach should be incorporated into HIV research pathway initiatives.

Viral and Host Mediators of Non-Suppressible HIV-1 Viremia.

Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

The Drug-Induced Interface That Drives HIV-1 Integrase Hypermultimerization and Loss of Function.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target. IMPORTANCE Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection.

Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.

Allosteric Integrase Inhibitor Influences on HIV-1 Integration and Roles of LEDGF/p75 and HDGFL2 Host Factors.

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.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.

Genome-wide CRISPR/Cas9 transcriptional activation screen identifies a histone acetyltransferase inhibitor complex as a regulator of HIV-1 integration.

The retrovirus human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. Although treatment of HIV/AIDS with antiretroviral therapy provides suppression of viremia, latent reservoirs of integrated proviruses preclude cure by current antiviral treatments. Understanding the mechanisms of host-viral interactions may elucidate new treatment strategies. Here, we performed a CRISPR/Cas9 transcriptional activation screen using a high-complexity, genome-wide sgRNA library to identify cellular factors that inhibit HIV-1 infection of human CD4+ T cells. MT4 cells were transduced with a CRISPR/Cas9 sgRNA library and infected with nef-deficient HIV-1NL4-3 expressing ganciclovir-sensitive thymidine kinase, thus enabling selection of HIV-1-resistant cells for analysis of enriched sgRNAs. After validation of screen hits, multiple host factors essential for HIV-1 infection were identified, including SET (SET nuclear proto-oncogene) and ANP32A (acidic nuclear phosphoprotein 32A, PP32A), which together form a histone acetylase inhibitor complex. Using multiple human cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-1-infected individuals, we demonstrate that SET depletion increased HIV-1 infectivity by augmenting DNA integration without significantly changing sites of integration. Conversely, SET overexpression decreased HIV-1 integration and infectivity. SET protein expression was significantly reduced in PBMCs from HIV-1-infected individuals and was downregulated by HIV-1 infection of healthy donor cells in vitro. Notably, HIV-1-induced downregulation of SET could be alleviated by inhibition of the protease granzyme A. Altogether, we have identified cellular inhibitors of HIV-1 infection on a genome-wide scale, which affords new insight into host-virus interactions and may provide new strategies for HIV-1 treatment.

Multimodal Functionalities of HIV-1 Integrase.

Integrase is the retroviral protein responsible for integrating reverse transcripts into cellular genomes. Co-packaged with viral RNA and reverse transcriptase into capsid-encased viral cores, human immunodeficiency virus 1 (HIV-1) integrase has long been implicated in reverse transcription and virion maturation. However, the underlying mechanisms of integrase in these non-catalytic-related viral replication steps have remained elusive. Recent results have shown that integrase binds genomic RNA in virions, and that mutational or pharmacological disruption of integrase-RNA binding yields eccentric virion particles with ribonucleoprotein complexes situated outside of the capsid shell. Such viruses are defective for reverse transcription due to preferential loss of integrase and viral RNA from infected target cells. Parallel research has revealed defective integrase-RNA binding and eccentric particle formation as common features of class II integrase mutant viruses, a phenotypic grouping of viruses that display defects at steps beyond integration. In light of these new findings, we propose three new subclasses of class II mutant viruses (a, b, and c), all of which are defective for integrase-RNA binding and particle morphogenesis, but differ based on distinct underlying mechanisms exhibited by the associated integrase mutant proteins. We also assess how these findings inform the role of integrase in HIV-1 particle maturation.