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Article ; Online: HIV eradication: combinatorial approaches to activate latent viruses.

De Crignis, Elisa / Mahmoudi, Tokameh

2014 Volume 6, Issue 11, Page(s) 4581–4608

Abstract: The concept of eradication of the Human Immune Deficiency Virus (HIV) from infected patients has gained much attention in the last few years. While combination Anti-Retroviral Therapy (c-ART) has been extremely effective in suppressing viral replication, ...

Abstract	The concept of eradication of the Human Immune Deficiency Virus (HIV) from infected patients has gained much attention in the last few years. While combination Anti-Retroviral Therapy (c-ART) has been extremely effective in suppressing viral replication, it is not curative. This is due to the presence of a reservoir of latent HIV infected cells, which persist in the presence of c-ART. Recently, pharmaceutical approaches have focused on the development of molecules able to induce HIV-1 replication from latently infected cells in order to render them susceptible to viral cytopathic effects and host immune responses. Alternative pathways and transcription complexes function to regulate the activity of the HIV promoter and might serve as molecular targets for compounds to activate latent HIV. A combined therapy coupling various depressors and activators will likely be the most effective in promoting HIV replication while avoiding pleiotropic effects at the cellular level. Moreover, in light of differences among HIV subtypes and variability in integration sites, the combination of multiple agents targeting multiple pathways will increase likelihood of therapeutic effectiveness and prevent mutational escape. This review provides an overview of the mechanisms that can be targeted to induce HIV activation focusing on potential combinatorial approaches.
MeSH term(s)	Drug Discovery/trends ; Drug Therapy, Combination/methods ; HIV Infections/drug therapy ; HIV Infections/virology ; HIV-1/drug effects ; HIV-1/physiology ; Humans ; Virus Latency/drug effects ; Virus Replication
Language	English
Publishing date	2014-11-21
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v6114581
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Article ; Online: Host Restriction Factors and Human Immunodeficiency Virus (HIV-1): A Dynamic Interplay Involving All Phases of the Viral Life Cycle.

D Urbano, Vanessa / De Crignis, Elisa / Re, Maria Carla

Current HIV research

2018 Volume 16, Issue 3, Page(s) 184–207

Abstract: Mammalian cells have evolved several mechanisms to prevent or block lentiviral infection and spread. Among the innate immune mechanisms, the signaling cascade triggered by type I interferon (IFN) plays a pivotal role in limiting the burden of HIV-1. In ... ...

Abstract	Mammalian cells have evolved several mechanisms to prevent or block lentiviral infection and spread. Among the innate immune mechanisms, the signaling cascade triggered by type I interferon (IFN) plays a pivotal role in limiting the burden of HIV-1. In the presence of IFN, human cells upregulate the expression of a number of genes, referred to as IFN-stimulated genes (ISGs), many of them acting as antiviral restriction factors (RFs). RFs are dominant proteins that target different essential steps of the viral cycle, thereby providing an early line of defense against the virus. The identification and characterization of RFs have provided unique insights into the molecular biology of HIV-1, further revealing the complex host-pathogen interplay that characterizes the infection. The presence of RFs drove viral evolution, forcing the virus to develop specific proteins to counteract their activity. The knowledge of the mechanisms that prevent viral infection and their viral counterparts may offer new insights to improve current antiviral strategies. This review provides an overview of the RFs targeting HIV-1 replication and the mechanisms that regulate their expression as well as their impact on viral replication and the clinical course of the disease.
MeSH term(s)	Gene Expression Regulation ; HIV-1/immunology ; HIV-1/physiology ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferon Type I/metabolism ; Signal Transduction ; Virus Replication
Chemical Substances	Interferon Type I
Language	English
Publishing date	2018-12-17
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2192348-6
ISSN	1873-4251 ; 1570-162X
ISSN (online)	1873-4251
ISSN	1570-162X
DOI	10.2174/1570162X16666180817115830
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Article ; Online: HIV Eradication

Elisa De Crignis / Tokameh Mahmoudi

Viruses, Vol 6, Iss 11, Pp 4581-

Combinatorial Approaches to Activate Latent Viruses

2014 Volume 4608

Abstract	The concept of eradication of the Human Immune Deficiency Virus (HIV) from infected patients has gained much attention in the last few years. While combination Anti-Retroviral Therapy (c-ART) has been extremely effective in suppressing viral replication, it is not curative. This is due to the presence of a reservoir of latent HIV infected cells, which persist in the presence of c-ART. Recently, pharmaceutical approaches have focused on the development of molecules able to induce HIV-1 replication from latently infected cells in order to render them susceptible to viral cytopathic effects and host immune responses. Alternative pathways and transcription complexes function to regulate the activity of the HIV promoter and might serve as molecular targets for compounds to activate latent HIV. A combined therapy coupling various depressors and activators will likely be the most effective in promoting HIV replication while avoiding pleiotropic effects at the cellular level. Moreover, in light of differences among HIV subtypes and variability in integration sites, the combination of multiple agents targeting multiple pathways will increase likelihood of therapeutic effectiveness and prevent mutational escape. This review provides an overview of the mechanisms that can be targeted to induce HIV activation focusing on potential combinatorial approaches.
Keywords	HIV latency ; latency reversing agents ; shock and kill strategy ; HIV transcription regulators ; Microbiology ; QR1-502 ; Science ; Q
Subject code	570
Language	English
Publishing date	2014-11-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
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Article ; Online: A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency.

Röling, Michael / Mollapour Sisakht, Mahsa / Ne, Enrico / Moulos, Panagiotis / Crespo, Raquel / Stoszko, Mateusz / De Crignis, Elisa / Bodmer, Helen / Kan, Tsung Wai / Akbarzadeh, Maryam / Harokopos, Vaggelis / Hatzis, Pantelis / Palstra, Robert-Jan / Mahmoudi, Tokameh

mBio

2021 Volume 12, Issue 6, Page(s) e0298021

Abstract: To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the ... ...

Abstract	To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4
MeSH term(s)	CD4-Positive T-Lymphocytes/metabolism ; CD4-Positive T-Lymphocytes/virology ; DNA Helicases/genetics ; DNA Helicases/metabolism ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Gene Expression Regulation, Viral ; HIV Infections/genetics ; HIV Infections/metabolism ; HIV Infections/virology ; HIV-1/genetics ; HIV-1/physiology ; Haploidy ; Host-Pathogen Interactions ; Humans ; Receptors, Kainic Acid/genetics ; Receptors, Kainic Acid/metabolism ; Trans-Activators/genetics ; Trans-Activators/metabolism ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Ubiquitin-Specific Proteases/genetics ; Ubiquitin-Specific Proteases/metabolism ; Virus Activation ; Virus Latency
Chemical Substances	DNA-Binding Proteins ; GRIK5 protein, human ; IRF2BP2 protein, human ; Receptors, Kainic Acid ; Trans-Activators ; Transcription Factors ; USP15 protein, human (EC 3.4.19.12) ; Ubiquitin-Specific Proteases (EC 3.4.19.12) ; DNA Helicases (EC 3.6.4.-) ; CHD9 protein, human (EC 3.6.4.12)
Language	English
Publishing date	2021-12-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2557172-2
ISSN	2150-7511 ; 2161-2129
ISSN (online)	2150-7511
ISSN	2161-2129
DOI	10.1128/mBio.02980-21
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Article ; Online: Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR.

Mancarella, Antonio / Procopio, Francesco A / Achsel, Tilmann / De Crignis, Elisa / Foley, Brian T / Corradin, Giampietro / Bagni, Claudia / Pantaleo, Giuseppe / Graziosi, Cecilia

Journal of visualized experiments : JoVE

2019 , Issue 153

Abstract: In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading ... ...

Abstract	In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.
MeSH term(s)	Adult ; HIV-1/genetics ; Humans ; Male ; Middle Aged ; Open Reading Frames/genetics ; RNA, Antisense/genetics ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Young Adult
Chemical Substances	RNA, Antisense
Language	English
Publishing date	2019-11-27
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/60511
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Article ; Online: Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).

Mancarella, Antonio / Procopio, Francesco A / Achsel, Tilmann / De Crignis, Elisa / Foley, Brian T / Corradin, Giampietro / Bagni, Claudia / Pantaleo, Giuseppe / Graziosi, Cecilia

The Journal of general virology

2019 Volume 100, Issue 5, Page(s) 863–876

Abstract: The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not ... ...

Abstract	The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.
MeSH term(s)	Adult ; Base Sequence/genetics ; CD4-Positive T-Lymphocytes/virology ; Gene Expression Regulation, Viral/genetics ; HIV Infections/virology ; HIV-1/genetics ; Humans ; Male ; Middle Aged ; Open Reading Frames/genetics ; RNA, Antisense/genetics ; RNA, Viral/genetics ; Virus Replication/genetics ; Young Adult
Chemical Substances	RNA, Antisense ; RNA, Viral
Language	English
Publishing date	2019-03-21
Publishing country	England
Document type	Journal Article
ZDB-ID	219316-4
ISSN	1465-2099 ; 0022-1317
ISSN (online)	1465-2099
ISSN	0022-1317
DOI	10.1099/jgv.0.001244
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Article: Restriction Factors expression decreases in HIV-1 patients after cART.

D'Urbano, Vanessa / Bertoldi, Alessia / Re, Maria Carla / De Crignis, Elisa / Tamburello, Martina / Primavera, Alessandra / Laginestra, Maria Antonella / Zanasi, Nicolò / Calza, Leonardo / Viale, Pier Luigi / Lazzarotto, Tiziana / Bon, Isabella / Gallinella, Giorgio

The new microbiologica

2021 Volume 44, Issue 2, Page(s) 95–103

Abstract: Activation of interferon (IFN) mediated responses and the consequent expression of restriction factors (RFs) represent an early line of defense against HIV-1 infection. The levels of viral replication and the antiviral are among the determinants ... ...

Abstract	Activation of interferon (IFN) mediated responses and the consequent expression of restriction factors (RFs) represent an early line of defense against HIV-1 infection. The levels of viral replication and the antiviral are among the determinants influencing RFs' expression pattern. A deeper understanding of the molecular mechanisms regulating RFs activity and their relationship with viral replication factors might lead to new therapeutic strategies based on the enhancement of immune response against the virus. The aim of this study is to perform a longitudinal evaluation of the variations in the levels of a group of selected RFs (APOBEC3G, BST2, TRIM5α, MX2, SAMHD1, SERINC3/5, IFI16 and STING) to determine the impact of cART on their expression in HIV-1 positive patients. Together with RFs expression, immunological and virological parameters (plasma HIV1-RNA load and total HIV1-DNA) were longitudinally evaluated in a cohorts fourteen HIV-1 cART na ve patients, who were evaluated at diagnosis (T0) and followed at 4 (T1) and 8 (T2) months after starting cART. Fourteen long-term treated patients who achieved sustained undetectable viremia for at least 2 years were also included in the study as a reference group. We observed a restoration of immunological conditions during cART, together with a progressive decrease of HIV1-RNA load, which became undetectable at 8 months after starting treatment. On the other hand, despite showing a trend towards decrease, total HIV1-DNA remained detectable after reaching viral suppression, similarly to what observed in long term treated patients. The expression of APOBEC3G, SAMHD1, BST2, IFI16, SERINC3, and SERINC5 was higher at the time of diagnosis and decreased significantly during therapy, reaching levels similar to the ones observed in virally suppressed patients. On the other hand, MX2 and TRIM5a high expression values up to T0, reaching lower levels immediately after the initiation of cART treatment. Correlation analysis showed a positive association between the expression levels of APOBEC3G, IFI16, MX2, SAMHD1, SERINC3 and TRIM5α with the HIV-1 viral load. On the contrary, no significant association was observed for BST2, SERINC5 and STING, even BST2 expression showed a tendency to correlate with viral load. We observed a tendency for a positive association of MX2, SAMHD1 and SERINC5 with the size of viral reservoir and a trend for a negative association for STING. STING appeared also as the only one factor whose expression correlates with the CD4 count and the CD4/CD8 ratio. Our data confirm the correlation between viral replication and expression of RFs, with, the levels of cellular defense proteins decreasing in parallel to the reduction of viral replication.
Language	English
Publishing date	2021-05-01
Publishing country	Italy
Document type	Journal Article
ZDB-ID	756168-4
ISSN	1121-7138 ; 0391-5352
ISSN	1121-7138 ; 0391-5352
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Article ; Online: Guidelines for the qualitative detection of viral genomes in dried blood spots.

Gibellini, Davide / De Crignis, Elisa / Re, Maria Carla

Methods in molecular biology (Clifton, N.J.)

2012 Volume 903, Page(s) 21–34

Abstract: Dried blood spots (DBSs) are a useful alternative to blood sampling especially in children or for screening high-risk populations in developing countries. DBS blood collection can be employed in the diagnosis of viral infections by PCR or RT-PCR and also ...

Abstract	Dried blood spots (DBSs) are a useful alternative to blood sampling especially in children or for screening high-risk populations in developing countries. DBS blood collection can be employed in the diagnosis of viral infections by PCR or RT-PCR and also in viral genome sequencing. In addition, the advent of multiplex PCR approaches has led to further diagnostic and methodological improvements allowing simultaneous detection of two or more different viral genomes in the same sample and amplification reaction. This chapter describes general guidelines for the qualitative viral genome amplification and detection in DBS providing an example application of a qualitative real-time SYBR Green-based multiplex RT-PCR assay targeting two major viral pathogens, HIV-1 and HCV.
MeSH term(s)	Blood Specimen Collection ; DNA, Viral/blood ; DNA, Viral/genetics ; DNA, Viral/isolation & purification ; Dried Blood Spot Testing/methods ; Genome, Viral/genetics ; Humans ; Molecular Diagnostic Techniques ; RNA, Viral/blood ; RNA, Viral/genetics ; RNA, Viral/isolation & purification ; Real-Time Polymerase Chain Reaction
Chemical Substances	DNA, Viral ; RNA, Viral
Language	English
Publishing date	2012
Publishing country	United States
Document type	Journal Article ; Practice Guideline ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-937-2_2
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Article ; Online: T cell toxicity of HIV latency reversing agents.

Zhao, Manzhi / De Crignis, Elisa / Rokx, Casper / Verbon, Annelies / van Gelder, Teun / Mahmoudi, Tokameh / Katsikis, Peter D / Mueller, Yvonne M

Pharmacological research

2018 Volume 139, Page(s) 524–534

Abstract: Combination antiretroviral therapy reduces morbidity and mortality in HIV infected patients. However, the cure of HIV infection is hindered by the persistence of the latent HIV reservoir. Latency reversing agents (LRAs) are developed to target the HIV ... ...

Abstract	Combination antiretroviral therapy reduces morbidity and mortality in HIV infected patients. However, the cure of HIV infection is hindered by the persistence of the latent HIV reservoir. Latency reversing agents (LRAs) are developed to target the HIV latently infected cells for HIV reactivation. In addition to reversal of HIV latency, the eradication of HIV latently infected cells will require effector HIV-specific CD8+ T cells. Therefore it is imperative we understand how LRAs affect immune cells. We have performed a comparative in depth analysis of the cytotoxicity of several compounds belonging to four LRA classes on T cells, B cells, and NK cells. In addition, the effect of these LRAs on activation and inhibitory receptor expression of CD8+ T cells was examined. We show that the HDAC inhibitors romidepsin and panobinostat are highly cytotoxic for CD4+ and CD8+ T cells, whereas the PKC agonists bryostatin and prostratin and BET inhibitors JQ1 and OXT-015 were less cytotoxic. The BAF inhibitors CAPE and pyrimethamine exhibit no cytotoxicity. Drug-specific cytotoxicity on CD8+ T cells was comparable between healthy controls and cART-treated HIV-infected patients. Bryostatin and both BET inhibitors downregulated the expression of CD279 on CD8+ T cells without affecting their activation. Our comparison of LRAs identified differences in cytotoxicity between LRA classes and members within a class and suggests that some LRAs such as bryostatin and BET inhibitors may also downregulate inhibitory receptors on activated HIV-specific CD8+ T cells. These findings may guide the use of LRAs that have the capacity to preserve or restore CD8+ T cell immunity.
MeSH term(s)	Anti-HIV Agents/pharmacology ; Cells, Cultured ; HIV Infections/drug therapy ; HIV-1/physiology ; Humans ; T-Lymphocytes/drug effects ; Virus Latency/drug effects
Chemical Substances	Anti-HIV Agents
Language	English
Publishing date	2018-10-23
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1003347-6
ISSN	1096-1186 ; 0031-6989 ; 1043-6618
ISSN (online)	1096-1186
ISSN	0031-6989 ; 1043-6618
DOI	10.1016/j.phrs.2018.10.023
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Article ; Online: Development of C-TILDA: A modified TILDA method for reservoir quantification in long term treated patients infected with subtype C HIV-1.

Bertoldi, Alessia / D'Urbano, Vanessa / Bon, Isabella / Verbon, Annelies / Rokx, Casper / Boucher, Charles / van Kampen, Jeroen J A / Gruters, Rob A / Gallinella, Giorgio / Calza, Leonardo / Mahmoudi, Tokameh / De Crignis, Elisa / Re, Maria Carla

Journal of virological methods

2019 Volume 276, Page(s) 113778

Abstract: A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting ... ...

Abstract	A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting dilution assay (TILDA) combines serial dilution of CD4
MeSH term(s)	Antiviral Agents/therapeutic use ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes/virology ; DNA, Viral/blood ; HIV/genetics ; HIV/isolation & purification ; HIV Infections/blood ; HIV Infections/drug therapy ; HIV Testing/methods ; Humans ; Sensitivity and Specificity ; Sustained Virologic Response ; Viral Load/methods ; Virus Latency
Chemical Substances	Antiviral Agents ; DNA, Viral
Language	English
Publishing date	2019-11-19
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2019.113778
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