scholarly journals Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus

2021 ◽  
Author(s):  
Sereina Olivia Sutter ◽  
Anouk Lkharrazi ◽  
Elisabeth Maria Schraner ◽  
Kevin Michaelsen ◽  
Anita Felicitas Meier ◽  
...  
Keyword(s):  
Single Cell ◽  
Virus Type ◽  
Helper Virus ◽  
Single Cell Level ◽  
Cellular Functions ◽  
Cell Level ◽  
Structural Reorganization ◽  
Adeno Associated Virus ◽  
Viral Genomes

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

scholarly journals Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

2007 ◽  
Vol 81 (8) ◽  
pp. 3721-3730 ◽  
Author(s):  
Mary Murphy ◽  
Janette Gomos-Klein ◽  
Marko Stankic ◽  
Erik Falck-Pedersen
Keyword(s):  
Virus Type ◽  
Transcriptional Control ◽  
Helper Virus ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Rep Proteins

ABSTRACT The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

scholarly journals The transcriptional characteristics of mast cells derived from skin tissue in type 2 diabetes patients at the single-cell level

2021 ◽  
Vol 123 (7) ◽  
pp. 151789
Author(s):  
Bingye Liao ◽  
Qiuyi Ouyang ◽  
Hongqin Song ◽  
Ziqi Wang ◽  
Jinhua Ou ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Mast Cells ◽  
Single Cell ◽  
Skin Tissue ◽  
Single Cell Level ◽  
Cell Level ◽  
Diabetes Patients

scholarly journals Herpes Simplex Virus Type 1 ICP0 Protein Mediates Activation of Adeno-Associated Virus Type 2 rep Gene Expression from a Latent Integrated Form

2004 ◽  
Vol 78 (20) ◽  
pp. 10977-10986 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Alberto L. Epstein ◽  
Estelle Toublanc ◽  
Philippe Moullier ◽  
Anna Salvetti
Keyword(s):  
Gene Expression ◽  
Herpes Simplex ◽  
Virus Type ◽  
Helper Virus ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Adeno-associated virus type 2 (AAV-2) is a human parvovirus that requires the presence of a helper virus, such as the herpes simplex virus type 1 (HSV-1) to accomplish a complete productive cycle. In the absence of helper virus, AAV-2 can establish a latent infection that is characterized by the absence of expression of viral genes. So far, four HSV-1 early genes, UL5/8/52 (helicase primase complex) and UL29 (single-stranded DNA-binding protein), were defined as sufficient for AAV replication when cells were transfected with a plasmid carrying the wild-type AAV-2 genome. However, none of these viral products was shown to behave as a transcriptional factor able to activate AAV gene expression. Our study provides the first evidence that the immediate-early HSV-1 protein ICP0 can promote rep gene expression in cells latently infected with wild-type AAV-2. This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Furthermore, using deletion mutants, we demonstrate that the localization of ICP0 to ND10 and their disruption is not required for the activation of the rep promoter, whereas binding of ICP0 to the ubiquitin-specific protease HAUSP makes a significant contribution to this effect.

scholarly journals Cell-to-Cell Variation in Defective Virus Expression and Effects on Host Responses during Influenza Virus Infection

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Chang Wang ◽  
Christian V. Forst ◽  
Tsui-wen Chou ◽  
Adam Geber ◽  
Minghui Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Single Cell ◽  
Influenza Virus Infection ◽  
Host Responses ◽  
Viral Transcription ◽  
Single Cell Level ◽  
Cell Level ◽  
Viral Genomes ◽  
Defective Virus

ABSTRACT Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection. IMPORTANCE Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species in vitro. This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.

scholarly journals Cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection

2018 ◽  
Author(s):  
Chang Wang ◽  
Christian V. Forst ◽  
Tsui-wen Chou ◽  
Adam Geber ◽  
Minghui Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Influenza A ◽  
Influenza Infection ◽  
Host Responses ◽  
Viral Transcription ◽  
Single Cell Level ◽  
Cell Level ◽  
Viral Genomes ◽  
Cell Variation ◽  
Defective Virus

ABSTRACTVirus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single cell level and how it impacts virus-host interactions at a systems level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability of viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show a correlation between DVGs and viral-induced variation of the host transcriptional program and an association between differential induction of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single cell level improve our understanding of the complex virus-host interplay during influenza infection.IMPORTANCEDefective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate disease severity and pathogenicity of the influenza infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single cell transcriptional profiling. Here we characterized virus and host transcriptomes in individual influenza-infected cells, including that of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective virus genome speciesin vitro. This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.

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