Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma.

OBJECTIVE: Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This study aimed to precisely characterise HBV integrations, in relation with viral and host genomics and clinical features. DESIGN: A novel pipeline was set up to perform viral capture on tumours and non-tumour liver tissues from a French cohort of 177 patients mainly of European and African origins. Clonality of each integration event was determined with the localisation, orientation and content of the integrated sequence. In three selected tumours, complex integrations were reconstructed using long-read sequencing or Bionano whole genome mapping. RESULTS: Replicating HBV DNA was more frequently detected in non-tumour tissues and associated with a higher number of non-clonal integrations. In HCC, clonal selection of HBV integrations was related to two different mechanisms involved in carcinogenesis. First, integration of viral enhancer nearby a cancer-driver gene may lead to a strong overexpression of oncogenes. Second, we identified frequent chromosome rearrangements at HBV integration sites leading to cancer-driver genes (TERT, TP53, MYC) alterations at distance. Moreover, HBV integrations have direct clinical implications as HCC with a high number of insertions develop in young patients and have a poor prognosis. CONCLUSION: Deep characterisation of HBV integrations in liver tissues highlights new HBV-associated driver mechanisms involved in hepatocarcinogenesis. HBV integrations have multiple direct oncogenic consequences that remain an important challenge for the follow-up of HBV-infected patients.

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration.

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration.

BACKGROUND: A protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication. RESULTS: Here we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication. CONCLUSIONS: These results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

Integrating molecular dynamics simulation and molecular mechanics/generalized Born surface area calculation into pharmacophore modeling: a case study on the proviral integration site for Moloney murine leukemia virus (Pim)-1 kinase inhibitors.

Communicated by Ramaswamy H. Sarma.

The Presence of Human Papillomavirus DNA Integration is Associated with Poor Clinical Results in Patients with Third-Stage Cervical Cancer.

The presence of virus DNA integration into the cell genome was studied for 47 primary HPV16-positive patients with morphologically verified stage III cervical cancer. By using ROC analysis, the patients were divided into two groups: with and without HPV DNA integration into the host cell genome. The differences between the groups by the histological type, degree of tumor differentiation, and primary response to therapy were statistically insignificant. Virus DNA integration more than 7-fold reduced 5-year relapse-free survival and 1.7-fold reduced overall survival rate in comparison with patients without HPV DNA integration (p=0.0002 and p=0.05, respectively). The relative risk of adverse outcome of the disease in patients with the presence of HPV16 DNA integration increases by 4 times over a period of less than 3 years (р=0.0006) at high AUC level. The probability of earlier progression of the disease in patients with of HPV DNA integration calculated according to the Cox proportional hazards model was 85.5% (hazard ratio 5.96; p=0.002). Thus, the results suggest that the presence of HPV16 DNA integration into the cell genome is an independent factor in predicting clinical outcome of advanced cervical cancer and can serve as an effective criterion for the individual choice of treatment tactics for the patients.

Three-Fragment Fluorescence Complementation for Imaging of Ternary Complexes under Physiological Conditions.

Protein-protein interactions (PPIs) occur in a vast variety of cellular processes, and many processes are regulated by multiple protein interactions. Identification of PPIs is essential for the analysis of biological pathways and to further understand underlying molecular mechanisms. However, visualization and identification of multiprotein complexes, including ternary complexes in living cells under physiological conditions, remains challenging. In this work, we reported a three-fragment fluorescence complementation (TFFC) by splitting the Venus fluorescent protein for visualizing ternary complexes in living cells under physiological conditions. With this Venus-based TFFC system, we identified the multi-interaction of weak-affinity ternary complexes under physiological conditions. The TFFC system was further applied to the analysis of multi-interactions during the HIV-1 integration process, revealing the important role of the barrier-to-autointegration factor protein in HIV-1 integration. This TFFC system provides a useful tool for visualizing and identifying ternary complexes in living cells under physiological conditions.

Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR.

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

Modulated expression of the HIV-1 2LTR zinc finger efficiently interferes with the HIV integration process.

Lentiviral vectors have emerged as the most efficient system to stably transfer and insert genes into cells. By adding a tetracycline (Tet)-inducible promoter, transgene expression delivered by a lentiviral vector can be expressed whenever needed and halted when necessary. Here we have constructed a doxycycline (Dox)-inducible lentiviral vector which efficiently introduces a designed zinc finger protein, 2-long terminal repeat zinc-finger protein (2LTRZFP), into hematopoietic cell lines and evaluated its expression in pluripotent stem cells. As a result this lentiviral inducible system can regulate 2LTRZFP expression in the SupT1 T-cell line and in pluripotent stem cells. Using this vector, no basal expression was detected in the T-cell line and its induction was achieved with low Dox concentrations. Remarkably, the intracellular regulatory expression of 2LTRZFP significantly inhibited HIV-1 integration and replication in HIV-inoculated SupT1 cells. This approach could provide a potential tool for gene therapy applications, which efficiently control and reduce the side effect of therapeutic genes expression.

HIV infection results in clonal expansions containing integrations within pathogenesis-related biological pathways.

The genomic integration of HIV into cells results in long-term persistence of virally infected cell populations. This integration event acts as a heritable mark that can be tracked to monitor infected cells that persist over time. Previous reports have documented clonal expansion in people and have linked them to proto-oncogenes; however, their significance or contribution to the latent reservoir has remained unclear. Here, we demonstrate that a directed pattern of clonal expansion occurs in vivo, specifically in gene pathways important for viral replication and persistence. These biological processes include cellular division, transcriptional regulation, RNA processing, and posttranslational modification pathways. This indicates preferential expansion when integration events occur within genes or biological pathways beneficial for HIV replication and persistence. Additionally, these expansions occur quickly during unsuppressed viral replication in vivo, reinforcing the importance of early intervention for individuals to limit reservoir seeding of clonally expanded HIV-infected cells.

The Retrovirus Capsid Core.

The retrovirus capsid core is a metastable structure that disassembles during the early phase of viral infection after membrane fusion. The core is intact and permeable to essential nucleotides during reverse transcription, but it undergoes disassembly for nuclear entry and genome integration. Increasing or decreasing the stability of the capsid core has a substantial negative impact on virus infectivity, which makes the core an attractive anti-viral target. The retrovirus capsid core also encounters a variety of virus- and organism-specific host cellular factors that promote or restrict viral replication. This review describes the structural elements fundamental to the formation and stability of the capsid core. The physical and chemical properties of the capsid core that are critical to its functional role in reverse transcription and interaction with host cellular factors are highlighted to emphasize areas of current research.

Oligomerization of Retrovirus Integrases.

Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.

Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis.

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.

Cotesia congregata Bracovirus Circles Encoding PTP and Ankyrin Genes Integrate into the DNA of Parasitized Manduca sexta Hemocytes.

Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as the template for the production of double-stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production, thereby impeding viral replication in caterpillar hosts during parasitism. Here, we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata Sequences sharing similarities with host integration motifs (HIMs) of Microplitis demolitor bracovirus (MdBV) circles involved in integration into DNA could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore, by using a primer extension capture method based on these HIMs and high-throughput sequencing, we could show that 8 out of 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM, indicating that an HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.IMPORTANCE The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in braconid parasitoid wasp parasitism success. Indeed, results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins within newly dividing cells of caterpillar hosts that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

Let me in: Control of HIV nuclear entry at the nuclear envelope.

The nuclear envelope is a physical barrier that isolates the cellular DNA from the rest of the cell, thereby limiting pathogen invasion. The Human Immunodeficiency Virus (HIV) has a remarkable ability to enter the nucleus of non-dividing target cells such as lymphocytes, macrophages and dendritic cells. While this step is critical for replication of the virus, it remains one of the less understood aspects of HIV infection. Here, we review the viral and host factors that favor or inhibit HIV entry into the nucleus, including the viral capsid, integrase, the central viral DNA flap, and the host proteins CPSF6, TNPO3, Nucleoporins, SUN1, SUN2, Cyclophilin A and MX2. We review recent perspectives on the mechanism of action of these factors, and formulate fundamental questions that remain. Overall, these findings deepen our understanding of HIV nuclear import and strengthen the favorable position of nuclear HIV entry for antiviral targeting.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression.

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression.

Retroviral intasomes arising.

Retroviral DNA integration takes place in the context of the intasome nucleoprotein complex. X-ray crystal structures of functional spumaviral intasomes were previously revealed to harbor a homotetramer of integrase, and it was generally believed that integrase tetramers catalyzed the integration of other retroviruses. The elucidation of new structures from four different retroviruses over the past year has however revealed this is not the case. The number of integrase molecules required to construct the conserved intasome core structure differs between viral species. While four subunits suffice for spumaviruses, α- and ß-retroviruses require eight and the lentiviruses use up to sixteen. Herein we described these alternative architectures, highlighting both evolutionary and structural constraints that result in the different integrase-DNA stoichiometries across Retroviridae.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. METHODS: E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. RESULTS: All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. CONCLUSION: HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.

Retrovirus Integration: Some Assembly Required?

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang et al., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription.