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Article ; Online: Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.

Shah, Raven / Lan, Shuiyun / Puray-Chavez, Maritza N / Liu, Dandan / Tedbury, Philip R / Sarafianos, Stefan G

Journal of visualized experiments : JoVE

2020 , Issue 164

Abstract: Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA ... ...

Abstract	Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.
MeSH term(s)	DNA, Viral/analysis ; DNA, Viral/genetics ; HIV-1/genetics ; Hepacivirus/genetics ; Hepatitis B virus/genetics ; Humans ; In Situ Hybridization, Fluorescence ; Optical Imaging ; Orthomyxoviridae/genetics ; RNA, Viral/analysis ; RNA, Viral/genetics ; Single-Cell Analysis ; Viral Proteins/analysis ; Virus Diseases/diagnosis ; Virus Diseases/genetics ; Zika Virus/genetics
Chemical Substances	DNA, Viral ; RNA, Viral ; Viral Proteins
Language	English
Publishing date	2020-10-29
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/61843
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication.

Puray-Chavez, Maritza N / Farghali, Mahmoud H / Yapo, Vincent / Huber, Andrew D / Liu, Dandan / Ndongwe, Tanyaradzwa P / Casey, Mary C / Laughlin, Thomas G / Hannink, Mark / Tedbury, Philip R / Sarafianos, Stefan G

Viruses

2019 Volume 11, Issue 7

Abstract: Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly ... ...

Abstract	Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA, and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines, providing strong evidence for the involvement of MOV10 in the HBV life cycle. We demonstrated that MOV10 does not interact with HBV-core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. We conclude that the restriction of HBV by MOV10 is mediated through effects at the level of viral RNA.
MeSH term(s)	Animals ; Cell Line ; Cells, Cultured ; Gene Expression Regulation, Viral ; Hepatitis B/virology ; Hepatitis B virus/physiology ; Host-Pathogen Interactions ; Humans ; Mice ; Microbial Interactions ; Moloney murine leukemia virus/physiology ; Protein Binding ; RNA ; RNA Helicases/metabolism ; RNA, Viral ; Viral Proteins/metabolism ; Virus Replication
Chemical Substances	RNA, Viral ; Viral Proteins ; pgRNA ; RNA (63231-63-0) ; Mov10 protein, human (EC 2.7.7.-) ; RNA Helicases (EC 3.6.4.13)
Language	English
Publishing date	2019-07-17
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v11070651
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

Viruses. 2019 July 17, v. 11, no. 7

2019

Abstract	Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA, and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines, providing strong evidence for the involvement of MOV10 in the HBV life cycle. We demonstrated that MOV10 does not interact with HBV-core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. We conclude that the restriction of HBV by MOV10 is mediated through effects at the level of viral RNA.
Keywords	CRISPR-Cas systems ; DNA ; Hepatitis B virus ; RNA helicases ; cell lines ; gene editing ; gene overexpression ; hepatitis B ; leukemia ; small interfering RNA ; virus replication ; viruses
Language	English
Dates of publication	2019-0717
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v11070651
Database	NAL-Catalogue (AGRICOLA)

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Article: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

Liu, Dandan / Tedbury, Philip R / Lan, Shuiyun / Huber, Andrew D / Puray-Chavez, Maritza N / Ji, Juan / Michailidis, Eleftherios / Saeed, Mohsan / Ndongwe, Tanyaradzwa P / Bassit, Leda C / Schinazi, Raymond F / Ralston, Robert / Rice, Charles M / Sarafianos, Stefan G

Viruses. 2019 Nov. 08, v. 11, no. 11

2019

Abstract: RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA (( ...

Abstract	RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.
Keywords	DNA ; Hepatitis C virus ; RNA ; antiviral agents ; confocal microscopy ; drugs ; fluorescence in situ hybridization ; hepatitis C ; image analysis ; mechanism of action ; pathogens ; viral proteins ; viruses
Language	English
Dates of publication	2019-1108
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v11111039
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus.

Viruses

2019 Volume 11, Issue 11

Abstract	RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.
MeSH term(s)	Antiviral Agents/pharmacology ; Cell Line ; Hepacivirus/drug effects ; Hepacivirus/genetics ; Hepatitis C/drug therapy ; Hepatitis C/virology ; Humans ; In Situ Hybridization, Fluorescence/methods ; RNA, Viral/drug effects ; RNA, Viral/metabolism ; Virus Replication/drug effects ; Zika Virus/drug effects ; Zika Virus/genetics ; Zika Virus Infection/drug therapy ; Zika Virus Infection/virology
Chemical Substances	Antiviral Agents ; RNA, Viral
Language	English
Publishing date	2019-11-08
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v11111039
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Novel Hepatitis B Virus Capsid-Targeting Antiviral That Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores.

Huber, Andrew D / Pineda, Dallas L / Liu, Dandan / Boschert, Kelsey N / Gres, Anna T / Wolf, Jennifer J / Coonrod, Emily M / Tang, Jing / Laughlin, Thomas G / Yang, Qiongying / Puray-Chavez, Maritza N / Ji, Juan / Singh, Kamalendra / Kirby, Karen A / Wang, Zhengqiang / Sarafianos, Stefan G

ACS infectious diseases

2019 Volume 5, Issue 5, Page(s) 750–758

Abstract: An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the ... ...

Abstract	An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.
MeSH term(s)	Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; Hep G2 Cells ; Hepatitis B virus/drug effects ; Hepatitis B virus/physiology ; Hepatocytes/drug effects ; Hepatocytes/virology ; Humans ; Viral Core Proteins/antagonists & inhibitors ; Virus Assembly/drug effects ; Virus Internalization/drug effects ; Virus Replication/drug effects
Chemical Substances	Antiviral Agents ; Viral Core Proteins
Language	English
Publishing date	2019-01-14
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2373-8227
ISSN (online)	2373-8227
DOI	10.1021/acsinfecdis.8b00235
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Article ; Online: The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells.

Huber, Andrew D / Wolf, Jennifer J / Liu, Dandan / Gres, Anna T / Tang, Jing / Boschert, Kelsey N / Puray-Chavez, Maritza N / Pineda, Dallas L / Laughlin, Thomas G / Coonrod, Emily M / Yang, Qiongying / Ji, Juan / Kirby, Karen A / Wang, Zhengqiang / Sarafianos, Stefan G

mSphere

2018 Volume 3, Issue 2

Abstract: Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid ... ...

Abstract	Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly
MeSH term(s)	Antiviral Agents/pharmacology ; Capsid/chemistry ; Capsid/drug effects ; Capsid Proteins/chemistry ; Capsid Proteins/genetics ; Fluorescent Antibody Technique ; Hep G2 Cells ; Hepatitis B/drug therapy ; Hepatitis B virus/drug effects ; Hepatitis B virus/physiology ; Humans ; Inclusion Bodies, Viral/chemistry ; Protein Aggregates/drug effects ; Pyridines/pharmacology ; Pyrimidines/pharmacology ; Recombinant Proteins/chemistry ; Virus Assembly/drug effects ; Virus Replication/drug effects
Chemical Substances	Antiviral Agents ; BAY 38-7690 ; Capsid Proteins ; Protein Aggregates ; Pyridines ; Pyrimidines ; Recombinant Proteins
Language	English
Publishing date	2018-04-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2379-5042
ISSN (online)	2379-5042
DOI	10.1128/mSphereDirect.00131-18
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Article ; Online: 3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H.

Huber, Andrew D / Michailidis, Eleftherios / Tang, Jing / Puray-Chavez, Maritza N / Boftsi, Maria / Wolf, Jennifer J / Boschert, Kelsey N / Sheridan, Megan A / Leslie, Maxwell D / Kirby, Karen A / Singh, Kamalendra / Mitsuya, Hiroaki / Parniak, Michael A / Wang, Zhengqiang / Sarafianos, Stefan G

Antimicrobial agents and chemotherapy

2017 Volume 61, Issue 6

Abstract: Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel ...

Abstract	Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range.
Language	English
Publishing date	2017-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	217602-6
ISSN	1098-6596 ; 0066-4804
ISSN (online)	1098-6596
ISSN	0066-4804
DOI	10.1128/AAC.00245-17
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Zs.A 809: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: SAMHD1 has differential impact on the efficacies of HIV nucleoside reverse transcriptase inhibitors.

Huber, Andrew D / Michailidis, Eleftherios / Schultz, Megan L / Ong, Yee T / Bloch, Nicolin / Puray-Chavez, Maritza N / Leslie, Maxwell D / Ji, Juan / Lucas, Anthony D / Kirby, Karen A / Landau, Nathaniel R / Sarafianos, Stefan G

Antimicrobial agents and chemotherapy

2014 Volume 58, Issue 8, Page(s) 4915–4919

Abstract: Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) ... ...

Abstract	Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.
MeSH term(s)	Adenine/analogs & derivatives ; Adenine/pharmacology ; Cell Line ; Dideoxynucleotides/metabolism ; Gene Expression ; Genes, Reporter ; HIV Reverse Transcriptase/antagonists & inhibitors ; HIV Reverse Transcriptase/genetics ; HIV Reverse Transcriptase/metabolism ; HIV-1/drug effects ; HIV-1/enzymology ; Host-Pathogen Interactions ; Humans ; Lamivudine/pharmacology ; Luciferases/genetics ; Luciferases/metabolism ; Monocytes/drug effects ; Monocytes/metabolism ; Monocytes/virology ; Monomeric GTP-Binding Proteins/antagonists & inhibitors ; Monomeric GTP-Binding Proteins/genetics ; Monomeric GTP-Binding Proteins/metabolism ; Organophosphonates/pharmacology ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; Reverse Transcriptase Inhibitors/pharmacology ; SAM Domain and HD Domain-Containing Protein 1 ; Stavudine/pharmacology ; Tenofovir ; Virus Replication/drug effects ; Zidovudine/pharmacology
Chemical Substances	Dideoxynucleotides ; Organophosphonates ; RNA, Small Interfering ; Reverse Transcriptase Inhibitors ; Lamivudine (2T8Q726O95) ; Zidovudine (4B9XT59T7S) ; Tenofovir (99YXE507IL) ; Stavudine (BO9LE4QFZF) ; Luciferases (EC 1.13.12.-) ; HIV Reverse Transcriptase (EC 2.7.7.49) ; SAM Domain and HD Domain-Containing Protein 1 (EC 3.1.5.-) ; SAMHD1 protein, human (EC 3.1.5.-) ; Monomeric GTP-Binding Proteins (EC 3.6.5.2) ; Adenine (JAC85A2161)
Language	English
Publishing date	2014-05-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	217602-6
ISSN	1098-6596 ; 0066-4804
ISSN (online)	1098-6596
ISSN	0066-4804
DOI	10.1128/AAC.02745-14
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