scholarly journals Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma

2021 ◽  
Vol 9 (3) ◽  
pp. 520
Author(s):  
Taro Ikegami ◽  
Hitoshi Hirakawa ◽  
Narutoshi Tsukahara ◽  
Akikazu Murakami ◽  
Norimoto Kise ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Dna ◽  
Laryngeal Papilloma ◽  
Functional Studies ◽  
Dna And Rna ◽  
Coordinated Expression ◽  
Molecular Therapies ◽  
Targeted Molecular Therapies

Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 105 ± 1.86 × 105 copies/ng DNA. E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.

scholarly journals Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2024
Author(s):  
Taro Ikegami ◽  
Norimoto Kise ◽  
Hidetoshi Kinjyo ◽  
Shunsuke Kondo ◽  
Mikio Suzuki ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Positive Reaction ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Laryngeal Papilloma ◽  
Hpv Dna ◽  
Functional Studies ◽  
Rna In Situ Hybridization

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

scholarly journals General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism

2011 ◽  
Vol 92 (12) ◽  
pp. 2757-2769 ◽  
Author(s):  
Martin Zydek ◽  
Ralf Uecker ◽  
Nina Tavalai ◽  
Thomas Stamminger ◽  
Christian Hagemeier ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Nuclear Genome ◽  
Viral Gene Expression ◽  
Lytic Replication ◽  
Immediate Early ◽  
Viral Gene ◽  
Level Control ◽  
G2 Phase

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36–38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

scholarly journals Gammaherpesvirus RNAs Come Full Circle

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Nathan A. Ungerleider ◽  
Scott A. Tibbetts ◽  
Rolf Renne ◽  
Erik K. Flemington
Keyword(s):  
Adaptive Immune Response ◽  
Noncoding Rnas ◽  
Viral Gene Expression ◽  
Circular Rna ◽  
Circular Rnas ◽  
Viral Gene ◽  
Functional Studies ◽  
Barr Virus ◽  
Epstein Barr ◽  
Gammaherpesvirus 68

ABSTRACTAfter an adaptive immune response is mounted, gammaherpesviruses achieve persistence through the utilization of viral noncoding RNAs to craft a suitable host cell environment in an immunologically transparent manner. While gammaherpesvirus long noncoding RNAs (lncRNAs) and microRNAs have been recognized for some time and have been actively investigated, a recent spate of reports have now identified repertoires of the circular RNA (circRNA) class of noncoding RNAs in both the lymphocryptovirus and rhadinovirus genera of gammaherpesviruses. Despite the recent nature of these findings, the detection of circRNAs across viruses and viral gene expression programs, the conservation of some viral circRNAs, and their detection in the clinical setting already raises the spectrum of functional importance in gammaherpesvirus biology and associated malignancies. Here, we provide an overview of currently known gammaherpesvirus circular RNAs and discuss reported physical and contextual properties that may be germane to future functional studies. With the Epstein-Barr virus (EBV) circRNAome being the most extensively studied to date, our discussions will be weighted toward EBV circRNAs while also addressing circRNAs discovered in the rhesus macaque lymphocryptovirus (rLCV), the Kaposi’s sarcoma herpesvirus (KSHV), and the murid gammaherpesvirus 68 (MHV68). We hope that this will help set the stage for future investigations into the functions and relevance of this new class of viral noncoding RNAs in infection and disease.

scholarly journals Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Benjamin A. Diner ◽  
Krystal K. Lum ◽  
Jared E. Toettcher ◽  
Ileana M. Cristea
Keyword(s):  
Gene Expression ◽  
Live Cell Imaging ◽  
Nuclear Periphery ◽  
Viral Gene Expression ◽  
Live Cell ◽  
Viral Gene ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTThe human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.IMPORTANCEHow mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.

scholarly journals The Cellular Protein Daxx Interacts with Avian Sarcoma Virus Integrase and Viral DNA To Repress Viral Transcription

2005 ◽  
Vol 79 (8) ◽  
pp. 4610-4618 ◽  
Author(s):  
James G. Greger ◽  
Richard A. Katz ◽  
Alexander M. Ishov ◽  
Gerd G. Maul ◽  
Anna Marie Skalka
Keyword(s):  
Gene Expression ◽  
Histone Deacetylases ◽  
Viral Gene Expression ◽  
Cellular Protein ◽  
Viral Gene ◽  
Viral Transcription ◽  
Avian Sarcoma Virus ◽  
Viral Dna ◽  
Sarcoma Virus ◽  
Avian Sarcoma

ABSTRACT The cellular protein Daxx was identified as an interactor with avian sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.

scholarly journals DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression

1999 ◽  
Vol 73 (7) ◽  
pp. 5757-5766 ◽  
Author(s):  
James Chambers ◽  
Ana Angulo ◽  
Dhammika Amaratunga ◽  
Hongqing Guo ◽  
Ying Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Dna Sequences ◽  
De Novo ◽  
Expression Profiles ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Protein Synthesis ◽  
Entire Genome

ABSTRACT We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (α), early (β), early-late (γ1), and late (γ2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5′ noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for β, γ1, and γ2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

scholarly journals Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Sung-Chan Wei ◽  
Chih-Hsuan Tsai ◽  
Wei-Ting Hsu ◽  
Yu-Chan Chao
Keyword(s):  
Immune System ◽  
Gene Activation ◽  
Viral Gene Expression ◽  
Vero Cells ◽  
Nuclear Body ◽  
Viral Gene ◽  
Death Domain ◽  
Chromosomal Dna ◽  
Viral Dna ◽  
Body Formation

ABSTRACTUpon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA and forms unique nuclear body structures, which we term clathrate cage-like apparatus (CCLAs), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy as an enclosed environment for proper viral gene expression.IMPORTANCEThe major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3, which are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation; our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.

scholarly journals Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

2015 ◽  
Vol 90 (1) ◽  
pp. 486-496 ◽  
Author(s):  
Xiuji Cui ◽  
Daniel N. Clark ◽  
Kuancheng Liu ◽  
Xiao-Dong Xu ◽  
Ju-Tao Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Innate Immune Response ◽  
Viral Gene Expression ◽  
Innate Immune ◽  
Viral Gene ◽  
Viral Dna ◽  
B Virus

ABSTRACTHepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication.IMPORTANCEChronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence.

Analysis of avian leukosis virus DNA and RNA in bursal tumors: Viral gene expression is not required for maintenance of the tumor state

Cell ◽  
1981 ◽  
Vol 23 (2) ◽  
pp. 311-322 ◽  
Author(s):  
Gregory S. Payne ◽  
Sara A. Courtneidge ◽  
Lyman B. Crittenden ◽  
Aly M. Fadly ◽  
J.Michael Bishop ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Gene Expression ◽  
Avian Leukosis Virus ◽  
Viral Gene ◽  
Dna And Rna
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