Publications by authors named "Marina Lusic"

40 Publications

HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells.

Elife 2021 Apr 27;10. Epub 2021 Apr 27.

Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.
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http://dx.doi.org/10.7554/eLife.64776DOI Listing
April 2021

Viruses in the Nucleus.

Cold Spring Harb Perspect Biol 2021 Mar 22. Epub 2021 Mar 22.

Department of Infectious Diseases, Integrative Virology, Heidelberg University Hospital and German Center for Infection Research, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany.

Viral infection is intrinsically linked to the capacity of the virus to generate progeny. Many DNA and some RNA viruses need to access the nuclear machinery and therefore transverse the nuclear envelope barrier through the nuclear pore complex. Viral genomes then become chromatinized either in their episomal form or upon integration into the host genome. Interactions with host DNA, transcription factors or nuclear bodies mediate their replication. Often interfering with nuclear functions, viruses use nuclear architecture to ensure persistent infections. Discovering these multiple modes of replication and persistence served in unraveling many important nuclear processes, such as nuclear trafficking, transcription, and splicing. Here, by using examples of DNA and RNA viral families, we portray the nucleus with the virus inside.
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http://dx.doi.org/10.1101/cshperspect.a039446DOI Listing
March 2021

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Cell 2021 Feb 10;184(4):1032-1046.e18. Epub 2021 Feb 10.

European Molecular Biology Laboratory, Structural and Computational Biology Unit, 69117 Heidelberg, Germany; Max Planck Institute of Biophysics, Department of Molecular Sociology, 60438 Frankfurt, Germany. Electronic address:

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.
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http://dx.doi.org/10.1016/j.cell.2021.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895898PMC
February 2021

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Bioessays 2021 03 30;43(3):e2000257. Epub 2020 Dec 30.

Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.
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http://dx.doi.org/10.1002/bies.202000257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883048PMC
March 2021

3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4 T Cells.

Methods Mol Biol 2021 ;2157:239-249

Department of Infectious Diseases, CIID, Integrative Virology, Heidelberg University Hospital and German Center for Infection Research, Heidelberg, Germany.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4 T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.
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http://dx.doi.org/10.1007/978-1-0716-0664-3_14DOI Listing
March 2021

Patient-Derived HIV-1 Nef Alleles Reveal Uncoupling of CD4 Downregulation and SERINC5 Antagonism Functions of the Viral Pathogenesis Factor.

J Acquir Immune Defic Syndr 2020 10;85(2):e23-e26

Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Heidelberg, Germany.

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http://dx.doi.org/10.1097/QAI.0000000000002418DOI Listing
October 2020

Spatial-Temporal Variations in Atmospheric Factors Contribute to SARS-CoV-2 Outbreak.

Viruses 2020 05 27;12(6). Epub 2020 May 27.

Heidelberg University Hospital, 69120 Heidelberg, Germany.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has reached over five million confirmed cases worldwide, and numbers are still growing at a fast rate. Despite the wide outbreak of the infection, a remarkable asymmetry is observed in the number of cases and in the distribution of the severity of the COVID-19 symptoms in patients with respect to the countries/regions. In the early stages of a new pathogen outbreak, it is critical to understand the dynamics of the infection transmission, in order to follow contagion over time and project the epidemiological situation in the near future. While it is possible to reason that observed variation in the number and severity of cases stems from the initial number of infected individuals, the difference in the testing policies and social aspects of community transmissions, the factors that could explain high discrepancy in areas with a similar level of healthcare still remain unknown. Here, we introduce a binary classifier based on an artificial neural network that can help in explaining those differences and that can be used to support the design of containment policies. We found that SARS-CoV-2 infection frequency positively correlates with particulate air pollutants, and specifically with particulate matter 2.5 (PM), while ozone gas is oppositely related with the number of infected individuals. We propose that atmospheric air pollutants could thus serve as surrogate markers to complement the infection outbreak anticipation.
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http://dx.doi.org/10.3390/v12060588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354543PMC
May 2020

Alterations of redox and iron metabolism accompany the development of HIV latency.

EMBO J 2020 05 11;39(9):e102209. Epub 2020 Mar 11.

Heidelberg University Hospital, Heidelberg, Germany.

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4 T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.
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http://dx.doi.org/10.15252/embj.2019102209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196916PMC
May 2020

NHC-gold compounds mediate immune suppression through induction of AHR-TGFβ1 signalling in vitro and in scurfy mice.

Commun Biol 2020 3;3:10. Epub 2020 Jan 3.

1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar -heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies showed that the lead compound (MC3) activates TGFβ1 signaling and suppresses CD4 T-cell activation in vitro, in human and mouse T cells. Conversely, genetic knockdown or chemical inhibition of AHR activity or of TGFβ1-SMAD-mediated signaling offsets the MC3-mediated immunosuppression. In scurfy mice, a mouse model of human immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, MC3 treatment reduced autoimmune phenotypes and extended lifespan from 24 to 58 days. Our findings suggest that the immunosuppressive activity of gold compounds can be improved by introducing planar NHC ligands to activate the AHR-associated immunosuppressive pathway, thus expanding their potential clinical application for autoimmune diseases.
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http://dx.doi.org/10.1038/s42003-019-0716-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941985PMC
January 2020

Multifunctional Roles of the N-Terminal Region of HIV-1Nef Are Mediated by Three Independent Protein Interaction Sites.

J Virol 2019 12 12;94(1). Epub 2019 Dec 12.

Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Heidelberg, Germany

HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the ef-ssociated inase omplex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the -Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1Nef to facilitate immune evasion and virus spread. HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.
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http://dx.doi.org/10.1128/JVI.01398-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912122PMC
December 2019

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

Nat Commun 2019 09 6;10(1):4059. Epub 2019 Sep 6.

Department of Infectious Diseases, Integrative Virology, Heidelberg University Hospital and German Center for Infection Research, Heidelberg, Germany.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4 T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.
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http://dx.doi.org/10.1038/s41467-019-12046-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731298PMC
September 2019

Navigating through the nucleus with a virus.

Curr Opin Genet Dev 2019 04 31;55:100-105. Epub 2019 Aug 31.

Heidelberg University Hospital, Heidelberg, 69120, Germany; German Center for Infection Research, Heidelberg, 69120, Germany. Electronic address:

In each cell, the hierarchical organisation of the ∼2m DNA fibre ensures different nuclear functions, particularly proper gene expression. Chromosomes are non-randomly positioned occupying specific chromosome territories in the 3D nuclear space and circumventing several nuclear landmarks the Nuclear Envelope with embedded Nuclear Pore Complexes, Splicing Speckles, PML bodies and many others. At a higher level of organisation, similarly regulated chromatin regions cluster together in so called Topologically Associated Domains, TADs, while on a smaller scale, DNA sequences wrapped around histones dictate binding of transcription factors or inhibitors that determine the level of chromatin compaction. As intracellular pathogens, viruses explore different cellular structures and functions to either promote their lytic infection or control the latent state of their replication cycles. Here we highlight the most recent discoveries on how different levels of nuclear architecture and genome are exploited by various human viruses.
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http://dx.doi.org/10.1016/j.gde.2019.07.001DOI Listing
April 2019

Potential impact of the antirheumatic agent auranofin on proviral HIV-1 DNA in individuals under intensified antiretroviral therapy: Results from a randomised clinical trial.

Int J Antimicrob Agents 2019 Nov 5;54(5):592-600. Epub 2019 Aug 5.

Department of Infectious Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy. Electronic address:

Antiretroviral therapy (ART) is typically composed of a combination of three antiretroviral drugs and is the treatment of choice for people with human immunodeficiency virus type 1/acquired immune deficiency syndrome (HIV-1/AIDS). However, it is unable to impact on viral reservoirs, which harbour latent HIV-1 genomes that are able to reignite the infection upon treatment suspension. The aim of this study was to provide an estimate of the safety of the disease-modifying antirheumatic agent auranofin and its impact on the HIV-1 reservoir in humans under intensified ART. For this purpose, an interim analysis was conducted of three of the six arms of the NCT02961829 clinical trial (five patients each) with: no intervention, i.e. continuation of first-line ART; intensified ART (ART + dolutegravir and maraviroc); and intensified ART plus auranofin. Auranofin treatment was found to be well tolerated. No major adverse events were detected apart from a transient decrease in CD4 T-cell counts at Weeks 8 and 12. Auranofin decreased total viral DNA in peripheral blood mononuclear cells compared with ART-only regimens at Week 20 (P = 0.036) and induced a decrease in integrated viral DNA as quantified by Alu PCR. Despite the limited number of patient-derived sequences available in this study, phylogenetic analyses of nef sequences support the idea that auranofin may impact on the viral reservoir. [ClinicalTrials.gov ID: NCT02961829].
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http://dx.doi.org/10.1016/j.ijantimicag.2019.08.001DOI Listing
November 2019

Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression.

Mol Cell Biol 2019 07 27;39(14). Epub 2019 Jun 27.

Schaller Research Group at CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany

Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.
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http://dx.doi.org/10.1128/MCB.00553-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597885PMC
July 2019

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Nat Commun 2019 02 25;10(1):926. Epub 2019 Feb 25.

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149, Trieste, Italy.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.
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http://dx.doi.org/10.1038/s41467-019-08810-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389893PMC
February 2019

HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex.

Elife 2019 01 23;8. Epub 2019 Jan 23.

Department of Infectious Diseases Virology, University of Heidelberg, Heidelberg, Germany.

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.
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http://dx.doi.org/10.7554/eLife.41800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400501PMC
January 2019

Coloring hidden viruses.

Authors:
Marina Lusic

Elife 2018 05 29;7. Epub 2018 May 29.

Center for Integrative Infectious Disease Research, University of Heidelberg, Heidelberg, Germany.

An improved dual-color reporter reveals how the fate of latent HIV-1 depends on where it integrates in the human genome.
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http://dx.doi.org/10.7554/eLife.37732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973827PMC
May 2018

Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na,K-ATPase Blocks HIV-1 Infection.

EBioMedicine 2017 Jul 13;21:170-181. Epub 2017 Jun 13.

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy. Electronic address:

Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na,K-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.
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http://dx.doi.org/10.1016/j.ebiom.2017.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514404PMC
July 2017

Corrigendum: Nuclear landscape of HIV-1 infection and integration.

Nat Rev Microbiol 2017 02 27. Epub 2017 Feb 27.

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http://dx.doi.org/10.1038/nrmicro.2017.22DOI Listing
February 2017

Nuclear landscape of HIV-1 infection and integration.

Nat Rev Microbiol 2017 02 12;15(2):69-82. Epub 2016 Dec 12.

Howard Hughes Medical Institute; Department of Medicine, Johns Hopkins University School of Medicine, Room 879, Edward D. Miller Research Building, 733 North Broadway, Baltimore, Maryland 21205, USA.

To complete its life cycle, HIV-1 enters the nucleus of the host cell as reverse-transcribed viral DNA. The nucleus is a complex environment, in which chromatin is organized to support different structural and functional aspects of cell physiology. As such, it represents a challenge for an incoming viral genome, which needs to be integrated into cellular DNA to ensure productive infection. Integration of the viral genome into host DNA depends on the enzymatic activity of HIV-1 integrase and involves different cellular factors that influence the selection of integration sites. The selection of integration site has functional consequences for viral transcription, which usually follows the integration event. However, in resting CD4 T cells, the viral genome can be silenced for long periods of time, which leads to the generation of a latent reservoir of quiescent integrated HIV-1 DNA. Integration represents the only nuclear event in the viral life cycle that can be pharmacologically targeted with current therapies, and the aspects that connect HIV-1 nuclear entry to HIV-1 integration and viral transcription are only beginning to be elucidated.
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http://dx.doi.org/10.1038/nrmicro.2016.162DOI Listing
February 2017

Connecting HIV-1 integration and transcription: a step toward new treatments.

FEBS Lett 2016 07 16;590(13):1927-39. Epub 2016 Jun 16.

Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg and German Center for Infection Research (DZIF), Germany.

Thanks to the current combined antiretroviral therapy (cART), HIV-1 infection has become a manageable although chronic disease. The reason for this lies in the fact that long-lived cellular reservoirs persist in patients on cART. Despite numerous efforts to understand molecular mechanisms that contribute to viral latency, the important question of how and when latency is established remains unanswered. Related to this is the connection between HIV-1 integration and the capacity of the provirus to enter the latent state. In this review, we will give an overview of these nuclear events in the viral life cycle in the light of current therapeutic approaches, which aim to either reactivate the provirus or even excise the proviral DNA from the cellular genome.
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http://dx.doi.org/10.1002/1873-3468.12226DOI Listing
July 2016

HIV-1 transcriptional silencing caused by TRIM22 inhibition of Sp1 binding to the viral promoter.

Retrovirology 2015 Dec 18;12:104. Epub 2015 Dec 18.

Viral Pathogens and Biosafety Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, P2-P3 Laboratories, DIBIT, Via Olgettina n.58, 20132, Milan, Italy.

Background: Intracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR.

Findings: 293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4(+) SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays.

Conclusion: TRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter.
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http://dx.doi.org/10.1186/s12977-015-0230-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683785PMC
December 2015

Nuclear architecture dictates HIV-1 integration site selection.

Nature 2015 May 2;521(7551):227-31. Epub 2015 Mar 2.

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.
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http://dx.doi.org/10.1038/nature14226DOI Listing
May 2015

Ground control to Major Tom: "prepare for HIV landing".

Cell Host Microbe 2014 Nov 12;16(5):557-9. Epub 2014 Nov 12.

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy. Electronic address:

Productive HIV-1 infection requires viral cDNA integration into the host genome, an event catalyzed by HIV-1 integrase. In this issue of Cell Host & Microbe, Demeulemeester et al. (2014) report the existence of natural integrase polymorphisms that retarget viral integration away from gene-dense regions and are associated with rapid disease progression.
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http://dx.doi.org/10.1016/j.chom.2014.10.012DOI Listing
November 2014

Epigenetic modification at Notch responsive promoters blunts efficacy of inducing notch pathway reactivation after myocardial infarction.

Circ Res 2014 Sep 11;115(7):636-49. Epub 2014 Aug 11.

From the Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy (G.F., C.C., M.L., V.M., M.D.F., L.Z., S.Z., M.G.); and Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy (C.C., M.D.F., M.G.); and Center for Translational Cardiology, Azienda Ospedaliero-Universitaria "Ospedali Riuniti di Trieste", Trieste, Italy (C.C., M.D.F., M.G.).

Rationale: The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury.

Objective: We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice.

Methods And Results: In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation.

Conclusions: Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.304517DOI Listing
September 2014

Regulation of HIV-1 latency by chromatin structure and nuclear architecture.

J Mol Biol 2015 Feb 27;427(3):688-94. Epub 2014 Jul 27.

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, 34149 Trieste, Italy. Electronic address:

Current antiretroviral therapies fail to cure HIV-1 (human immunodeficiency virus type 1) infection because HIV-1 persists as a transcriptionally inactive provirus in resting memory CD4(+) T cells. Multiple molecular events are known to regulate HIV-1 gene expression, yet the mechanisms governing the establishment and maintenance of latency remain incompletely understood. Here we summarize different molecular aspects of viral latency, from its establishment in resting CD4(+) T cells to the mechanisms involved in the reactivation of latent viral reservoirs. We focus on the relevance of chromatin structure and nuclear architecture in determining the transcriptional fate of integrated HIV-1 genomes, in light of recent findings indicating that proximity to specific subnuclear neighborhoods regulates HIV-1 gene expression.
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http://dx.doi.org/10.1016/j.jmb.2014.07.022DOI Listing
February 2015

HIV-1 integrase binding to its cellular partners: a perspective from computational biology.

Curr Pharm Des 2014 ;20(21):3412-21

Computational Biophysics, German Research School for Simulation Sciences, Computational Biomedicine, Institute for Advanced Simulation (IAS-5), Forschungszentrum Jülich, Jülich D-52425, Germany.

Viral DNA integration into the infected cell genome is an essential step in the HIV-1 life cycle. Hence, the viral integrase enzyme has become an important target for antiviral therapy. The integrase's activity action relies on the binding to its cellular partners, therefore the knowledge of the structural determinants is very important from a therapeutic perspective. Here we first review published computer-aided structural predictions of HIV-1 integrase in complex with its interactors. These include DNA and the human HAT protein. Next, we present a prediction of the complex between HIV-1 integrase with the human prolyl-isomerase-1 (hPin1) enzyme. Interaction with hPin1 is crucial for efficient HIV-1 infection and it increases integrase stability (Manganaro et. al 2010, Nat. Med. 16, 329). The modeling presented here, which is validated against experimental data, provides a rationale for a variety of viral protein's mutations which impair protein function and HIV-1 virus replication in vivo without significantly affecting enzymatic activity.
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http://dx.doi.org/10.2174/13816128113199990631DOI Listing
March 2015

Proximity to PML nuclear bodies regulates HIV-1 latency in CD4+ T cells.

Cell Host Microbe 2013 Jun;13(6):665-77

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology ICGEB, 34149 Trieste, Italy.

Nuclear bodies (NBs), characterized by the presence of the promyelocytic leukemia (PML) protein, are important components of the nuclear architecture, contributing to genetic and epigenetic control of gene expression. In investigating the mechanisms mediating HIV-1 latency, we determined that silenced but transcriptionally competent HIV-1 proviruses reside in close proximity to PML NBs and that this association inhibits HIV-1 gene expression. PML binds to the latent HIV-1 promoter, which coincides with transcriptionally inactive facultative heterochromatic marks, notably H3K9me2, at the viral genome. PML degradation and NB disruption result in strong activation of viral transcription as well as release of G9a, the major methyltransferase responsible for H3K9me2, and loss of facultative heterochromatin marks from the proviral DNA. Additionally, HIV-1 transcriptional activation requires proviral displacement from PML NBs by active nuclear actin polymerization. Thus, nuclear topology and active gene movement mediate HIV-1 transcriptional regulation and have implications for controlling HIV-1 latency and eradication.
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http://dx.doi.org/10.1016/j.chom.2013.05.006DOI Listing
June 2013

Negative regulation of HIV-1 transcription by a heterodimeric NF-κB1/p50 and C-terminally truncated STAT5 complex.

J Mol Biol 2011 Jul;410(5):933-43

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milano, Italy.

Signal transducers and activator of transcription (STAT) proteins are often constitutively activated in leukocytes of HIV-1(+) individuals, which frequently show a dominant expression of a C-terminally truncated isoform of STAT5 (STAT5Δ). STAT5Δ can act as a negative regulator of human immunodeficiency virus type 1 (HIV-1) expression in both CD8-depleted primary leukocytes and chronically infected promonocytic U1 cells stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated STAT5Δ can directly bind to two consensus sequences in the HIV-1 long terminal repeat (LTR) promoter; binding impairs recruitment of RNA polymerase II (Crotti, A., Lusic, M., Lupo, R., Lievens, P. M., Liboi, E., Della Chiara, G., et al. (2007). Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression. Blood, 109, 5380-5389). One of the STAT consensus sequences overlaps with one nuclear factor κB (NF-κB) binding site; interestingly, NF-κB1/p50 homodimers, frequently detected in monocytic cells, are negative regulators of HIV transcription. Here, we show that GM-CSF stimulation of U1 cells, while not inducing NF-κB activation, leads to STAT5Δ phosphorylation and binding to the NF-κB/STAT target sequence in the HIV LTR promoter, which already associates with p50 under unstimulated conditions. STAT5Δ was found to associate with p50, but not with RelA/p65, in both U1 cells expressing endogenous proteins and 293T cells overexpressing these factors. Furthermore, GM-CSF stimulation promoted concurrent binding of STAT5Δ and p50 at the HIV LTR promoter in U1 cells. Immunoprecipitation of chromatin from GM-CSF-stimulated U1 cells confirmed in vivo binding of p50 to the viral promoter together with STAT5Δ. Thus, cytokine-activated STAT5Δ/p50 complexes can contribute to the maintenance of HIV-1 latency in monocytic cells.
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http://dx.doi.org/10.1016/j.jmb.2011.03.044DOI Listing
July 2011

The TRIM family protein KAP1 inhibits HIV-1 integration.

Cell Host Microbe 2011 Jun;9(6):484-95

Molecular Biology Laboratory, Scuola Normale Superiore, Piazza dei Cavalieri 7, Pisa 56126, Italy.

The integration of viral cDNA into the host genome is a critical step in the life cycle of HIV-1. This step is catalyzed by integrase (IN), a viral enzyme that is positively regulated by acetylation via the cellular histone acetyl transferase (HAT) p300. To investigate the relevance of IN acetylation, we searched for cellular proteins that selectively bind acetylated IN and identified KAP1, a protein belonging to the TRIM family of antiviral proteins. KAP1 binds acetylated IN and induces its deacetylation through the formation of a protein complex which includes the deacetylase HDAC1. Modulation of intracellular KAP1 levels in different cell types including T cells, the primary HIV-1 target, revealed that KAP1 curtails viral infectivity by selectively affecting HIV-1 integration. This study identifies KAP1 as a cellular factor restricting HIV-1 infection and underscores the relevance of IN acetylation as a crucial step in the viral infectious cycle.
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http://dx.doi.org/10.1016/j.chom.2011.05.004DOI Listing
June 2011