Search

Background The human myxovirus resistance 2 (Mx2/MxB) protein was originally found to regulate cytoplasmic-nuclear transport but was recently reported to restrict HIV-1 replication by binding to HIV-1 capsid (CA), preventing uncoating, the nuclear import of pre-integration complex (PIC) and viral DNA integration. This work explores the mechanisms of MxB-mediated HIV-1 inhibition. Results We demonstrated that MxB represses NUP358-mediated PIC nuclear import and HIV-1 replication. Moreover, MxB’s effects on PIC nuclear import and HIV-1 replication depend critically on cofactor cleavage and polyadenylation specificity factor subunit 6 (CPSF6). MxB binds nucleoporin NUP358, blocks NUP358-CA interaction, thereby impeding the nuclear import of HIV-1 PIC with CPSF6 binding to PIC. More intriguingly, CPSF6’s role in nuclear import depends on MxB, being a facilitator of HIV-1 nuclear import on its own, but becoming an inhibitor when MxB is present. Conclusions Our work establishes that MxB impedes the NUP358-mediated HIV-1 nuclear import and viral replication cooperatively with CPSF6.

How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of ∼100 nm, largely exceeding that of the NPC channel (∼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures., Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. In particular, for the HIV-1 genome to enter the nucleus, it must be led through the nuclear pore complex (NPC). During the HIV-1 cytoplasmic journey, the viral core acts as a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core is reshaped into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated with the preintegration complex (PIC) during and after nuclear translocation is still enigmatic. In this study, we analyzed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immunogold labeling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables the specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby offering the opportunity to uncover the architecture of the potential HIV-1 PIC. Thus, we combined the immunoelectron microscopy and ANCHOR technologies to reveal the presence of DNA- and CA-positive complexes by correlated light and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodeled in a pearl necklace-like shape. Thus, we could describe how CA proteins are reshaped around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed of the integrase and the retrotranscribed DNA leads the HIV-1 genome inside the host nucleus. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. Of note, we are now able to visualize the viral DNA in viral complexes, opening up new perspectives for future studies on virus’s fate in the cell nucleus. IMPORTANCE How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of ∼100 nm, largely exceeding that of the NPC channel (∼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

HIV-1 integration favors active chromatin, which is primarily mediated through interactions between the viral capsid and integrase proteins with host factors cleavage and polyadenylation specificity factor 6 (CPSF6) and lens epithelium-derived growth factor/p75, respectively. Previously published image-based studies had suggested that HIV-1 prefers to integrate into chromatin that associates spatially with the nuclear periphery. Here, we re-evaluated previously reported HIV-1 nuclear distance measures across studies and show that HIV-1 prefers peri-nuclear and mid-nuclear zones similarly, with a common preference between studies mapping to the boundary between these two radial areas. We also discuss emerging roles for the capsid-CPSF6 interaction in facilitating HIV-1 pre-integration complex nuclear import and subsequent intranuclear trafficking to preferred sites of viral DNA integration.

Nuclear entry of HIV-1 replication complexes through intact nuclear pore complexes is critical for successful infection. The host protein cleavage-and-polyadenylation-specificity-factor-6 (CPSF6) has been implicated in different stages of early HIV-1 replication. Applying quantitative microscopy of HIV-1 reverse-transcription and pre-integration-complexes (RTC/PIC), we show that CPSF6 is strongly recruited to nuclear replication complexes but absent from cytoplasmic RTC/PIC in primary human macrophages. Depletion of CPSF6 or lack of CPSF6 binding led to accumulation of HIV-1 subviral complexes at the nuclear envelope of macrophages and reduced infectivity. Two-color stimulated-emission-depletion microscopy indicated that under these circumstances HIV-1 complexes are retained inside the nuclear pore and undergo CA-multimer dependent CPSF6 clustering adjacent to the nuclear basket. We propose that nuclear entry of HIV-1 subviral complexes in macrophages is mediated by consecutive binding of Nup153 and CPSF6 to the hexameric CA lattice., eLife digest Viruses are miniscule parasites that hijack the resources of a cell to make more of themselves. For many, this involves getting inside the nucleus, the fortress that protects the cell’s genetic information. To do so, viruses need to first find a way through a double-layered membrane called the nuclear envelope, which only opens up when a cell divides. Yet, the human immunodeficiency virus type 1 (HIV-1) can infect cells that no longer divide, and in which the nucleus’ walls never come down. The virus cores then head for the nuclear pores, heavily guarded holes in the nuclear envelope that allow the cell's own molecules to go in and out of the nucleus. But HIV-1 is too big to fit through, as its genetic information is encased in a capsid, a coat made of a complex assembly of proteins. However, research shows that these capsid proteins can bind to host proteins at the pore or even inside the nucleus. For example, the capsid protein can recognize the pore protein Nup153, or the nuclear protein CPSF6. These interactions could help the virus make its way in, but how these events unfold is still unclear. To explore this, Bejarano, Peng et al. attached fluorescent labels to HIV-1 and watched as it infected non-dividing cells. Rather than completely get rid of their capsid before they crossed the pores, the virus particles hung on to a large part of their lattice. This remaining coat then attached to CPSF6; when this protein was missing or could not bind to capsid proteins, the viral complexes got stuck in the nuclear pores. This suggests that the capsid lattice could first interact with Nup153 inside the pores: then, CPSF6 would take over, knocking Nup153 away and pulling HIV-1 into the nucleus. Armed with this knowledge, virologists and drug developers could try to block HIV-1 from entering the cell’s nucleus; they could also start to dissect how drugs that target the HIV-1 capsid work. Ultimately, HIV-1 may serve as a model to unravel how large objects can pass the nuclear pore, which may help us understand how molecules are constantly trafficked in and out of the nucleus.

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes, however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.

Recognition of viral epitopes by the host immune system plays a pivotal role in controlling viral infection, while at the same time exerting selective pressure for escape mutations, which in turn leads to many epitopes evolving faster than the adjacent regions. Some epitope regions appear to be highly conserved despite the strong immune pressure due to functional and/or structural constraints acting on them. Yet, we still have relatively little understanding of the nature of protein structural and functional constraints operating on epitope regions. Here, we identify coevolving epitope regions that have high functional and/or structural constraints. We examined patterns of coevolution of protein segment pairs between Integrase-Reverse Transcriptase, Integrase-Vpr, and Reverse Transcriptase-Vpr protein interactions pairs in HIV-1 pre-integration complex. Our coevolutionary and structural analysis shows that protein regions with strong multiple coevolutionary constraints with few regions are located in structurally conserved regions (i.e., those with well-defined secondary structures). Meanwhile, protein regions with strong multiple coevolutionary constraints with many regions are clustered in the structurally flexible regions (regions with high B-factor) of the proteins and regions with high conformational diversity (relative mobility in the collective dynamics). On the other hand, protein regions with weak coevolutionary signals are clustered in structurally disordered regions. Identifying viral epitope regions that harbor strong constraints against escape mutations is important in development of vaccines that can induce immune responses against multiple conserved epitopes. This analysis also offers important insights into molecular evolution of protein interactions.

The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and vice versa, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration., Author summary In addition to matrix, capsid and nucleocapsid, the Gag polyproteins of many retroviruses also encode additional cleavage products, such as the p12 protein of murine leukemia virus. p12 is essential for both early and late replication events, but its function during early infection remains poorly characterised. Recent work has shown that the N-terminal domain of p12 binds to and stabilises the capsid shell that surrounds the viral core. Defects in the C-terminal domain of p12 prevented the viral pre-integration complex (PIC) from localising to chromatin during infection, and this could be rescued by the addition of a heterologous chromatin binding domain. In this study, we show that p12 is able to tether viral PICs containing capsid to mitotic chromatin by directly binding both hexameric capsid and nucleosomal histone proteins. Additionally, the affinity of p12 for chromatin may be enhanced by its binding to capsid and by its phosphorylation in mitosis. Excitingly, the mechanism of nucleosome binding appears to be conserved between gammaretroviruses and spumaretroviruses. Furthermore, influencing global nuclear targeting of the PIC by linking capsid to chromatin is reminiscent of the proposed role of the capsid-binding host protein, CPSF6, in HIV-1 infection.

Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation. Two examples of challenging protein families are described, namely, the human steroid nuclear receptors and the HIV-1 pre-integration complexes.

Transportin 3 (TNPO3) is a member of the importin-ß superfamily proteins. Despite numerous studies, the exact molecular mechanism of TNPO3 in retroviral infection is still controversial. Here, we provide evidence for the role and mechanism of TNPO3 in the replication of prototype foamy virus (PFV). Our findings revealed that PFV infection was reduced 2-fold by knockdown (KD) of TNPO3. However, late stage of viral replication including transcription, translation, viral assembly, and release was not influenced. The differential cellular localization of PFV integrase (IN) in KD cells pinpointed a remarkable reduction of viral replication at the nuclear import step. We also found that TNPO3 interacted with PFV IN but not with Gag, suggesting that IN-TNPO3 interaction is important for nuclear import of PFV pre-integration complex. Our report enlightens the mechanism of PFV interaction with TNPO3 and support ongoing research on PFV as a promising safe vector for gene therapy.

The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data to gain a better understanding of the mechanisms of HIV protein interactions.

Current treatments against the Aquired immune deficiency syndrome (AIDS) are reasonably effective in reducing the amount of human immunodeficiency virus (HIV) present in infected patients, but their side-effects, and the emergence of drug-resistant HIV strains have intensified the renewed search for novel anti-HIV therapies. An essential step in HIV infection is the integration of the viral genome into the host cell chromosomes within the nucleus. Unlike other retroviruses, HIV can transport its genetic material, in the form of the large nucleoprotein pre-integration complex (PIC), into the nucleus through the intact nuclear envelope (NE). This enables HIV to infect non-dividing cells such as macrophages and microglial cells. Detailed knowledge of the signal-dependent pathways by which cellular proteins and RNAs cross the NE has accumulated in the past decade, but although several different components of the PIC have been implicated in its nuclear import, the mechanism of nuclear entry remains unclear. Since specifically inhibiting PIC nuclear import would undoubtedly block HIV infection in non-dividing cells, this critical step of HIV replication is of great interest as a drug target. This review examines the complex and controversial literature regarding three PIC components - the HIV proteins matrix, integrase and Vpr - proposed to facilitate PIC nuclear import, and existing models of HIV PIC nuclear import. It also suggests approaches to move towards a better understanding of PIC nuclear import, through examining the role of individual PIC components in the context of the intact PIC by direct visualisation, in order to develop new anti-HIV therapeutics.

While Highly Active Antiretroviral Therapy (HAART) has significantly decreased the mortality of human immunodeficiency virus (HIV)-infected patients, emerging drug resistance to approved HIV-1 integrase inhibitors highlights the need to develop new antivirals with novel mechanisms of action. In this study, we screened a library of microbial natural compounds from endophytic fungus Colletotrichum sp. and identified alternariol 5-O-methyl ether (AME) as a compound that inhibits HIV-1 pre-integration steps. Time-of addition analysis, quantitative real-time PCR, confocal microscopy, and WT viral replication assay were used to elucidate the mechanism. As opposed to the approved integrase inhibitor Raltegravir, AME reduced both the integrated viral DNA and the 2-long terminal repeat (2-LTR) circular DNA, which suggests that AME impairs the nuclear import of viral DNA. Further confocal microscopy studies showed that AME specifically blocks the nuclear import of HIV-1 integrase and pre-integration complex without any adverse effects on the importin α/β and importin β-mediated nuclear import pathway in general. Importantly, AME inhibited Raltegravir-resistant HIV-1 strains and exhibited a broad anti-HIV-1 activity in diverse cell lines. These data collectively demonstrate the potential of AME for further development into a new HIV inhibitor, and suggest the utility of viral DNA nuclear import as a target for anti-HIV drug discovery.

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

We have investigated the organization and function of human immunodeficiency virus type 1 (HIV-1) preintegration complexes (PICs), the large nucleoprotein particles that carry out cDNA integration in vivo. PICs can be isolated from HIV-1-infected cells, and such particles are capable of carrying out integration reactions in vitro. We find that although the PICs are large, the cDNA must be condensed to fit into the measured volume. The ends of the cDNA are probably linked by a protein bridge, since coordinated joining of the two ends is not disrupted by cleaving the cDNA internally with a restriction enzyme. cDNA ends in PICs were protected from digestion by added exonucleases, probably due to binding of proteins. The intervening cDNA, in contrast, was susceptible to attack by endonucleases. Previous work has established that the virus-encoded integrase protein is present in PICs, and we have reported recently that the host protein HMG I(Y) is also present. Here we report that the viral matrix and reverse transcriptase (RT) proteins also cofractionated with PICs through several steps whereas capsid and nucleocapsid proteins dissociated. These data support a model of PIC organization in which the cDNA is condensed in a partially disassembled remnant of the viral core, with proteins tightly associated at the apposed cDNA ends but loosely associated with the intervening cDNA. In characterizing the structure of the cDNA ends, we found that the U5 DNA ends created by RT were ragged, probably due to the terminal transferase activity of RT. Only molecules correctly cleaved by integrase protein at the 3' ends were competent to integrate, suggesting that one role for terminal cleavage by integrase may be to create a defined end at otherwise heterogeneous cDNA termini.

Background To replicate their genome retroviruses need two key enzymes, the viral reverse transcriptase (RT) and integrase (IN). Soon after viral infection of a target cell, the genomic RNA is reverse transcribed by RT to generate the double-stranded viral DNA that interacts with viral and cellular proteins to form the pre-integration complex (PIC). IN is a key component of the PIC and is involved in several steps of retrovirus replication notably in reverse transcription, nuclear import, chromatin targeting and integration. IN cannot perform these functions on its own and need to recruit host cell proteins to efficiently carry out the different processes. Retroviral INs are proteins showing high inter-domain flexibility which accounts for their ability to interact with multiple partners, which in turn chaperone PIC formation and its multiple functions in integration. We assume that the different functions of the PIC in viral infection reflect the different conformations of their protein components as well as post-translational modifications. Biochemical and structural studies of the PIC have long been hampered due to the dynamics of composition and the intrinsic flexibility its components such as IN. We demonstrated that the low solubility and inter-domain flexibility can be circumvented by forming stable and specific complexes with DNA or protein co-factors and by post-translational modifications.

The early events of retroviral replication following virus–cell fusion and preceding integration of the viral genome into cellular chromatin occur within a context of a large multimolecular complex termed reverse transcription or preintegration complex. While some of the functions of these complexes (e.g., reverse transcription) are well described, most other events involved in assembly and processing of reverse transcription complexes, such as viral uncoating and nuclear transport, are poorly understood, and available data are very controversial. This article reviews the current literature on structure and function of reverse transcription complexes, focusing on the least known and most controversial steps.

International audience; HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.

Human immunodeficiency virus (HIV) infection yields a high level of non-integrated viral DNA in the infected cells and up to 99% of total viral DNA can be capable of transcription. This capability of non-integrated viral DNA is reducing the efficacy of anti-HIV drug development approaches that solely focus on the integration reactions of viral replication. Using kinetic modeling, we show the transient coordinated regulation of viral DNA production by retrovirus-encoded interacting enzymes reverse transcriptase (RT) and integrase (IN), and thus we identify a possible mechanism for reducing overall efficiency of viral replication. The results indicate that both IN·RT and IN·DNA complexes and their formation rates affect RT processivity and thereby the viral DNA expression level within the pre-integration complex.

Background HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood. Results Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization. Conclusions Our findings indicate that, shortly after viral entry, a significant portion of DNA–free IN that is distinct from active pre-integration complexes accumulates in the nucleus.

Nuclear import is a critical step in the life cycle of HIV-1. During the early (preintegration) stages of infection, HIV-1 has to transport its preintegration complex into the nucleus for integration into the host cell chromatin, while at the later (postintegration) stages viral regulatory proteins Tat and Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclear export of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1 life cycle, this step presents an attractive target for antiviral therapeutic intervention. In this review, we describe the current state of our understanding of the interactions regulating nuclear import of the HIV-1 preintegration complex and describe current approaches to inhibit it. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Although recent progress in highly active antiretroviral therapy (HAART) has provided an effective way to treat AIDS patients, the emergence of drug-resistant HIV-1 strains and drug toxicity during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the nuclear import process of the HIV pre-integration complex (PIC). The ability of HIV-1 using host cell nuclear import machinery to translocate the viral PIC into the cell nucleus is the critical determinant in the replication of the virus in non-dividing cells, such as macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for novel anti-HIV development. In this review, we will describe the mechanisms of HIV-1 PIC translocation into the nucleus and the structure-function of the viral and cellular factors involved in this process, as well as several classes of novel anti-HIV compounds which target the nuclear import of HIV-1 PIC and effectively block viral replication.

AIDS is the result of infection by a lentivirus, HIV-1, which primarily infects CD4+ T cells and macrophages. There is presently no vaccine and none will be available in the foreseeable future. Highly active antiretroviral drug therapy has led to a dramatic reduction of viral load in many infected individuals, and has decreased mortality in the developing world. However, besides long-term drug toxicity and eventual emergence of drug-resistant strains, withdrawal from the therapy (even after effective and continuous treatment) results in re-emergence of the virus since cells harbouring the latent viral reservoirs persist. These issues highlight the need for alternative therapies, e.g. gene therapy. This review summarizes various gene therapy strategies that target early stages of HIV-1 life cycle. We will cover strategies that allow interference at the level of the released virion RNA, reverse transcriptase, pre-integration complex, integrase, dsDNA and provirus DNA in gene-modified cells.

Virion infectivity factor (Vif) facilitates HIV infection by counteracting APOBEC3G late in replication in virus-producer cells. Here, we show that early after infection of new target cells Vif is part of the HIV reverse transcription machinery and acts as an accessory factor for reverse transcription. Vif protein was present in gradient fractions containing reverse transcription complexes (RTCs), and anti-Vif antibody immunoprecipitated HIV reverse transcription products from these gradient fractions. To investigate a role for Vif in RTCs independent of APOBEC3G, we created an intracellular environment that would restrict reverse transcription by pre-treating permissive target cells with 5-Fluoro 2-deoxyuridine, a thymidylate synthetase inhibitor, prior to infection with virus from permissive cells. Infectivity assays and quantitation of reverse transcription products demonstrated that replication of HIV lacking Vif was inhibited to a greater degree than wild type, without concurrent mutation of reverse transcription products, suggesting compromised reverse transcription in the absence of Vif.

The human protein EED belongs to the Polycomb Group. It acts as a transcriptional repressor and as a gene silencer. EED seems also to be important during the HIV-1 replication cycle, participating in the trafficking of preintegration complex and favouring the IN-mediated DNA integration reaction. EED has been overexpressed and purified in order to be crystallized alone or in complex with its viral partners IN, MA and Nef. A 3D structure model of EED has been obtained by homology modelling. Interaction regions determined by phage-display are localized on loops of the model.The psychrophilic P. haloplanktis produces the cellulase Cel5G. Its crystallographic structure was solved alone and in complex with cellobiose by molecular replacement at 1.4 Å and 1.6 Å resolution, respectively. Cellulase Cel5A from the mesophilic E. chrysanthemi has been used as a search model. Structural details and comparisons give new insights into the understanding of aspects governing the cold adaptation of the enzyme at a molecular level., La protéine humaine EED appartient à la famille des «Polycomb». Elle intervient dans la régulation génique. Elle jouerait aussi un rôle dans le cycle du virus de l'immunodéficience humaine (VIH-1) en participant au transport du complexe de préintégration et en facilitant la réaction d'intégration. EED a été surproduite afin d'être cristallisée seule et en complexe avec les protéines virales IN, MA et Nef. Un modèle de EED a été construit par homologie structurale. D'après nos expériences de « phage display », les régions susceptibles d'interagir avec les partenaires viraux se situent sur des boucles de ce modèle.La bactérie psychrophile P. haloplanktis produit la cellulase Cel5G. Les structures de cette cellulase seule et en complexe avec le cellobiose, ont été déterminées à haute résolution par remplacement moléculaire. La cellulase Cel5A de la bactérie mésophile E. chrysanthemi a été utilisée comme modèle. Nos résultats permettent de mieux comprendre les déterminants structuraux responsables de l'adaptation des enzymes aux basses températures.

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8·5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.

Replication of human immunodeficiency virus type 1 (HIV‐1) in non‐dividing cells critically depends on import of the viral pre‐integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV‐1 pre‐integration complexes in digitonin‐permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV‐1 pre‐integration complexes that lack Vpr failed to be imported in vitro , whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr − mutant was rescued when recombinant Vpr was re‐added. In addition, import of Vpr − virus was rescued by adding the cytosol of HeLa cells, where HIV‐1 replication had been shown to be Vpr‐independent. In a solution binding assay, Vpr associated with karyopherin α, a cellular receptor for NLSs. This association increased the affinity of karyopherin α for basic‐type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV‐1 pre‐integration complex and BSA–NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre‐integration complex to, and across, the nuclear membrane.

The p12 protein of the murine leukemia virus (MLV) is a constituent of the pre-integration complex (PIC) but its function in this complex remains unknown. We developed an imaging system to monitor MLV PIC trafficking in live cells. This allowed the visualization of PIC docking to mitotic chromosomes and its release upon exit from mitosis. Docking occurred concomitantly with nuclear envelope breakdown and was impaired for PICs of viruses with lethal p12 mutations. Insertion of a heterologous chromatin binding module into p12 of one of these mutants restored PICs attachment to the chromosomes and partially rescued virus replication. Capsid dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants. Altogether, these results explain, in part, MLV restriction to dividing cells and reveal a role for p12 as a factor that tethers MLV PIC to mitotic chromosomes., Author Summary Retroviruses, including the murine leukemia virus (MLV), reverse transcribe their RNA genome to a DNA copy, which travels from the cytoplasm to the nucleus as part of a ‘pre-integration complex’ (PIC), to integrate into cellular chromosomes. The viral p12 protein is a constituent of the MLV PIC, but its function in this complex has remained unknown. We developed a real-time imaging system to detect p12 and MLV PICs in live cells. This revealed that p12 tethers the MLV PIC to mitotic chromosomes. Accordingly, PICs derived from viruses with specific lethal mutations in p12 failed to attach to the chromosomes, and insertion of a heterologous chromatin binding module into p12 restored PICs attachment to the chromosomes and rescued virus replication. In addition, docking of wild type PICs to chromosomes coincided with nuclear envelope breakdown during mitosis, and detachment occurred upon exit from mitosis. Capsid - another viral component of the PIC - dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants, suggesting interplay between these two proteins in regulating targeting of mitotic chromosomes by the PIC. These results highlight steps contributing to the high tropism of MLV to dividing cells.

Virally encoded integrase (IN) proteins perform several important steps in the life cycle of retroviruses. During the early events of viral replication the RNA genome is converted into its cDNA copy which upon interaction with cellular and viral proteins generates the pre-integration complex (PIC). IN is a permanent component of the PIC. The Integrase Interactor Protein 1 (INI1) a homolog of yeast SNF5 and the lens epithelial derived growth factor (LEDGF) have been shown to interact with HIV-1 IN. In order to understand the mechanisms of the INI1 - mediated inhibition and/or activation functions in the early stage of HIV-1 infection, we analyzed the structure-function relationships of a quaternary complex comprising the full length wild type HIV-1 IN, the full length wild-type LEDGF, the INI1 IN binding domain (173-290) and viral U5 DNA. The stoichiometry of the components is 4/2/2/2, as shown by mass spectrometry and FCS. We determined for the first time the binding constants of U5 vDNA for IN by fluorescence anisotropy and found that the dissociation constants of IN/LEDGF and IN/LEDGF/INI1 for U5 vDNA remained in the same order of magnitude, while INI1 when bound to the IN/LEDGF complex inhibited the 3’processing reaction. CryoEM and in vitro functional analysis show that INI1, located within the cellular DNA binding site, inhibits the 3’ processing but not specific viral DNA binding. INI1 stabilizes the highly flexible integrase in a nonproductive conformation. Taken together, our data suggest that the role of INI1 could be to stabilize the highly flexible IN protein in a conformation that prevent non-specific interaction and auto integration during nucleosome targeting. Our results provide the basis for a novel type of integrase inhibitors (conformational inhibitors).

We have examined components of the preintegration complex of human immunodeficiency virus type 1 (HIV-1) and have analyzed features which govern the association of these components. HIV-1 nucleoprotein complexes, isolated from nuclear and cytoplasmic extracts of CD4+ cells after acute virus infection, contained viral RNA and DNA in association with viral matrix (MA), integrase (IN), and reverse transcriptase (RT) antigens but not capsid (CA) antigens and possessed integration activity in vitro. Association of IN but not RT or MA antigens with viral DNA was detergent-stable. Analysis of viral DNA synthesis and nuclear import of viral nucleoprotein complexes in the presence of a reversible RT inhibitor demonstrated that reverse transcription of viral RNA could be completed entirely in the host cell nucleus. Our studies demonstrate structural and functional features of the nucleoprotein (preintegration) complex of HIV-1 which are pertinent to the understanding of early events in the lentiviral life cycle.

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration., Author Summary All retroviruses reverse transcribe their RNA genome to a DNA copy in the cytoplasm of the infected cell. To be expressed, the viral genomic DNA has to travel to the cell nucleus and to integrate into the cellular chromosomes. This trafficking is governed by cellular and viral proteins that associate with the viral genome to form a ‘pre-integration complex’ (PIC), yet the full composition of this complex is unknown. Former studies showed that for the murine leukemia virus (MLV), mutations in a viral protein named p12 abrogate MLV infection, after reverse transcription and prior to the integration step, suggesting a role for this protein in early stages of infection. However, the precise mechanism of p12 action is not known. We combined microscopic, genetic and biochemical techniques to provide evidence that the p12 protein is part of the MLV PIC and that it exerts its function from within this complex. These analyses also suggest a role for p12 in the trafficking of the PIC from the cytoplasm to the chromosomes of the infected cell. Altogether, these findings highlight an important ‘building block’ of a complex that is essential for MLV infection.

The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle. To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al. 1987). The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro. Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally. The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions.

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.