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Ginkgo biloba, known for its distinctive fan-shaped leaves, has been used in traditional medicine for centuries, particularly in Asia. Ginkgo biloba is often touted as an herb for brain health, such as improving memory and cognition. This reputation does a great disservice to the most versatile herb in the world. Ginkgo biloba can be used as a potent antiviral agent for a variety of viruses. Ginkgo biloba has some key bioactive antiviral components: Flavonoids and Terpenoids: Ginkgo leaves contain high levels of flavonoids and terpenoids, compounds known for their antioxidant properties. These substances contribute to the antiviral activity of the plant. Ginkgolides and Bilobalides: These are unique terpene trilactones found in Ginkgo biloba, which have specific antiviral activities. Ginkgo Biloba's Mechanisms of Antiviral Action The antiviral properties of Ginkgo biloba are multi-faceted, involving multiple mechanisms: Inhibition of the fusions and synthesis of proteins in the viruses herpes simplex 1 and 2 (HSV-1 and HSV-2). Inhibition of genome replication in cytomegalovirus (HCMV) and Zika virus (ZIKV). Inhibition of viral fusion proteins in HIV, Ebola virus (EBOV), influenza A virus (IAV), and Epstein-Barr virus (EBV). Inhibition of the targeting protein and DNA of coronoviruses (SARS-CoV-2), varicella zoster virus (VZV), and measles virus. Inhibition of Viral Entry and Replication: Some studies suggest that Ginkgo biloba extracts can interfere with the ability of viruses to enter host cells or replicate. This is a key step in preventing the spread of viral infections. Immune System Modulation: Ginkgo biloba might enhance the body's immune response against viral infections. By modulating immune functions, it could help in controlling viral spread and severity. Anti-inflammatory Effects: The anti-inflammatory properties of Ginkgo biloba can be beneficial in reducing the severity of symptoms associated with viral infections. Research on Ginkgo Biloba's Antiviral Properties Anti-MERS-CoV and Anti-HCoV-229E Properties: A study focused on the antiviral activities of Ginkgo biloba leaf extracts against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Human Coronavirus 229E (HCoV-229E). Inhibition of Enveloped Viruses: Research published in Scientific Reports discussed how ginkgolic acid, a component of Ginkgo biloba, inhibits the fusion of enveloped viruses. The study found that ginkgolic acid had a strong inhibitory effect on Human Cytomegalovirus (HCMV) and also tested its effects on Herpes Simplex Virus-1 (HSV-1) and Zika Virus (ZIKV). Researchers also found broad spectrum inhibition by ginkgolic acid of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, inhibition was found of a non-enveloped adenovirus. The authors conclude that ginkolic acids may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)). It was observed that ginkgolic acid could inhibit the entry of these viruses into cells, thereby blocking viral replication. Another study entitled, “Ginkgolic acids inhibit SARS-CoV-2 and its variants by blocking the spike protein/ACE2 interplay” found that ginkgolic acids from ginkgo biloba inhibited the SARS-CoV-2 virus from binding to the ACE2 receptor and thus could potentially be helpful in acute COVID-19 infections. I use VascuSelect from Moss Nutrition which contains 120 mg of ginkgo biloba, 100 mg of grape seed extract, and 100 mg of mango fruit powder per capsule for acute and chronic viruses dosed 1 capsule once or twice a day with or without food. Grape seed extract and mango fruit powder have also been shown to have antiviral properties. This trio of herbs not only has antiviral prop...
Klaus Früh visits the Incubator to discuss his career and his work on cytomegalovirus-vectored vaccines which are unique in their ability to persistently maintain an immune shield of effector memory T cells, including highly unconventional MHC-II and MHC-E restricted CD8+ T cells. Host: Vincent Racaniello, Rich Condit, and Brianne Barker Guest: Klaus Früh Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode MicrobeTV Discord Server MicrobeTV store at Cafepress Spike shirts at vaccinated.us (promo code Microbetv) Research assistant position in Rosenfeld Lab CBER/FDA (pdf) HCMV-based attenuated vaccine platform (Sci Rep) HLA-E-restricted, Gag-specific CD8+ T cells suppress HIV-1 infection (Sci Immunol) RhCMV/SIV vaccine shows long-term efficacy against SIV challenge (Sci Transl Med) Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.03.531045v1?rss=1 Authors: Adelman, J. W., Rosas, S., Schumacher, M., Mokry, R., Terhune, S. S., Ebert, A. Abstract: Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that typically presents asymptomatically in healthy individuals despite lifelong latency. However, in 10-15% of congenital cases, this beta-herpesvirus demonstrates direct effects on the central nervous system, including microcephaly, cognitive/learning delays, and hearing deficits. HCMV has been widely shown to infect neural progenitor cells, but the permissiveness of fully differentiated neurons to HCMV is controversial and chronically understudied, despite potential associations between HCMV infection with neurodegenerative conditions. Using a model system representative of the human forebrain, we demonstrate that induced pluripotent stem cell (iPSC)-derived, excitatory glutamatergic and inhibitory GABAergic neurons are fully permissive to HCMV, demonstrating complete viral replication, competent virion production, and spread within the culture. Interestingly, while cell proliferation was not induced in these post-mitotic neurons, HCMV did increase expression of proliferative markers Ki67 and PCNA suggesting alterations in cell cycle machinery. These finding are consistent with previous HCMV-mediated changes in various cell types and implicate the ability of the virus to alter proliferative pathways to promote virion production. HCMV also induces significant structural changes in forebrain neurons, such as the formation of syncytia and retraction of neurites. Finally, we demonstrate that HCMV disrupts calcium signaling and decreases neurotransmission, with action potential generation effectively silenced after 15 days post infection. Taken together, our data highlight the potential for forebrain neurons to be permissive to HCMV infection in the CNS, which could have significant implications on overall brain health and function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Dr. Felicia Goodrum, Ph.D. ( https://profiles.arizona.edu/person/fgoodrum ) is Interim Associate Department Head and Professor of Immunobiology, as well as Professor, BIO5 Institute, Cellular and Molecular Medicine, Molecular and Cellular Biology, Cancer Biology And Genetics Graduate Interdisciplinary Programs, at the University of Arizona. Dr. Goodrum earned her Ph.D. from Wake Forest University School of Medicine studying cell cycle restrictions to adenovirus replication and then trained as a postdoctoral fellow at Princeton University in the laboratory of Dr. Thomas Shenk studying human cytomegalovirus latency. Dr. Goodrum joined the faculty at the University of Arizona in 2006, and her long-standing research focus is to understand the molecular virus-host interactions important to human cytomegalovirus (CMV) latency and persistence in the host. She has focused on identifying viral and host determinants mediating the switch between latent and replicative states. The goal of her research program is to define the mechanistic underpinnings of HCMV latency and reactivation to lay the foundation for clinical interventions to control CMV disease in all settings. Dr. Goodrum is the recipient of the Howard Temin Award from the National Cancer Institute, the Pew Scholar in Biomedical Sciences Award, and the Presidential Award for Early Career Scientists and Engineers. Support the show
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.02.530870v1?rss=1 Authors: O'Brien, B. S., Mokry, R., Schumacher, M., Rosas, S., Terhune, S. S., Ebert, A. Abstract: Human cytomegalovirus (HCMV) is a beta herpesvirus that causes severe congenital birth defects including microcephaly, vision loss, and hearing loss. Infection of cerebral organoids with HCMV causes significant downregulation of genes involved in critical neurodevelopmental pathways. The precise features of the infection causing this dysregulation remain unknown. Entry of HCMV into human cells is determined by the composition of glycoproteins in viral particles, which is influenced by the source of the virus. This includes a trimer complex and a pentamer complex with the latter enriched from replication in epithelial cells. To begin dissecting which features contribute to neuronal pathogenesis, we evaluated infection using virus from different sources along with the distribution of cellular entry receptors on cells in cerebral organoids. We observed significant increases in the number of viral genomes, viral spread and penetrance, and multinucleated syncytia in neural tissues infected with HCMV propagated in epithelial cells compared to fibroblasts. To determine if this was related to entry receptor distribution, we measured expressions of cellular entry receptors and observed similar distributions of all receptors on cells obtained from organoids indicating that source of virus is likely the key determinant. Next, we asked whether we could limit pathogenesis using neutralization antibodies. We found that pre-treatment with antibodies against viral glycoprotein B (gB) and gH successfully decreased viral genome levels, viral gene expression, and virus-induced syncytia. In contrast, targeting specific cellular entry receptors failed to limit infection. Using an antibody against gB, we also observed partial protection of developmental gene expression that was further improved by the addition of brain derived neurotropic factor (BDNF). These studies indicate that source of HCMV is a key determinant of neuronal pathogenesis that can be limited by neutralization antibodies and neurotropic factors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Hi! My name is Meiven Yang, and I am a rising junior at the University of Arizona majoring in Bioinformatics and Biochemistry. I have been working in Dr. Goodrum's lab since Summer of 2021, where we study Human Cytomegalovirus (HCMV). HCMV contributes to mortality among the immunocompromised and birth defects among newborns; there are currently limited therapies and no existing vaccines against the virus. My project focuses on mapping protein-protein interactions relative to the UL136 gene, which contributes to the regulation of viral latency and reactivation. After graduation, I am hoping to either work in industry or attend graduate school. During my free time, I enjoy spending time with family and friends, working out, and painting. Processed with VSCO with m5 preset
The TWiV crew reveal a unique portal on the calcivirus capsid formed upon receptor engagement, and the regulation of interferon responses in virus-infected cells by methylation of mRNA. Hosts: Vincent Racaniello, Dickson Despommier, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode European Congress of Virology 2019 ASM Clinical Virology Symposium Intel ISEF judges needed Calicivirus portal visualized (Nature) Calicivirus portal - long version (biorXiv) m6A modification and innate response (Nat Immunol) Brianne Barker's immunology course m6A modification and innate response (Genes Dev) Letters read on TWiV 534 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - The Red Queen's Race Card Game Alan- Pentax Papilio II Dickson- Felice Frankel on improving visual side of science Kathy- History of the alphabet chart (free download) usefulcharts.com (see blog, such as Latin Greek Cyrillic Venn Diagram) Vincent - Virology Lectures 2019 Listener Pick Peter- Microbehunter (YouTube, blog, forum) Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv
The TWiV crew reveal a unique portal on the calcivirus capsid formed upon receptor engagement, and the regulation of interferon responses in virus-infected cells by methylation of mRNA. Hosts: Vincent Racaniello, Dickson Despommier, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode European Congress of Virology 2019 ASM Clinical Virology Symposium Intel ISEF judges needed Calicivirus portal visualized (Nature) Calicivirus portal - long version (biorXiv) m6A modification and innate response (Nat Immunol) Brianne Barker's immunology course m6A modification and innate response (Genes Dev) Letters read on TWiV 534 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - The Red Queen's Race Card Game Alan- Pentax Papilio II Dickson- Felice Frankel on improving visual side of science Kathy- History of the alphabet chart (free download) usefulcharts.com (see blog, such as Latin Greek Cyrillic Venn Diagram) Vincent - Virology Lectures 2019 Listener Pick Peter- Microbehunter (YouTube, blog, forum) Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv
Vincent travels to Penn State College of Medicine in Hershey, PA to speak with Nick and Leslie about their careers and their work on human cytomegalovirus and retroviruses. Host: Vincent Racaniello Guests: Nicholas Buchkovich and Leslie Parent Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode HIV-1 gag membrane binding domain (J Virol) Nuclear experience for gag (PNAS) Gag in nucleolus (J Virol) YB-1 for retrovirus assembly (J Virol) HCMV controls BiP levels (J Virol) HCMV regulates La (J Virol) Inhibition of HCMV by modulating syntaxin 5 (J Virol) Non-envelopment role for ESCRT-III during HCMV infection (J Virol) Late retroviral budding function (J Virol) Grit by Angela Duckworth The Scientists by John Gribbin Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv
In episode 5 we talk with Dr Mike Weekes, a clinical consultant and Wellcome Trust Senior Clinical Fellow at the University of Cambridge about his lab’s work on using a technique he pioneered called ‘quantitative temporal viromics’ (which is a kind of proteomics). The Weekes lab uses this method to understand infection by human cytomegalovirus (HCMV – also known as human herpes virus 5) with an overall aim of discovering novel antiviral targets, which are required urgently. Image from Weekes et al., Cell. 2014 Jun 5;157(6):1460-72. doi: 10.1016/j.cell.2014.04.028. Edited intro/outro music credit: 'Take me higher' by 'Jahzzar'. freemusicarchive.org/music/Jahzzar/…Me_Higher_1626 (creativecommons.org/licenses/by-sa/4.0/).
Human cytomegalovirus (HCMV) is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs), the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs) give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs). Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A) showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb), TRL345, reactive with glycoprotein B (gB), but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.
Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy.
Background: A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). Results: Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer. Conclusions: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.
Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.
A virally-encoded G protein-coupled receptor promotes cellular proliferation and vascularization.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06
The M94 gene of murine cytomegalovirus (MCMV) is one of the about 40 core genes of the subfamily of β-Herpesvirinae with unknown function. MCMV is utilised as an in vivo model for studying human cytomegalovirus (HCMV) due to the strict species specifity of HCMV and major homologies to MCMV. HCMV is an important human pathogen with worldwide distribution. Although infection of the immune competent host is usually clinically silent, infection of the foetus and the newborn causes severe organ damage and infection of immunocompromised patients induces life-threatening disease. M94 has not been studied intensely as a single gene before, but studies on its homologues in other Herpesviridae demonstrated expression as true late gene, presence in the virion, interaction of UL94 and UL99 in HCMV and capsid binding in α-Herpesvirinae. While UL94 in HCMV is essential, M94 homologues in α-Herpesvirinae are not essential, indicating an essential function of M94 conserved in β-Herpesvirinae but lost in α-Herpesvirinae. For the identification of this essential function, a library of random mutants was generated by a modified Tn7 transposon mutagenesis. The modifications in the transposon mutagenesis produced a library of high diversity and wide coverage containing 32,000 primary clones including stop and insertion mutants. The analysis of 613 clones by sequencing resulted in 399 unique mutants containing a correct 15 base pair (bp) insertion in M94. 116 stop and insertion mutants were individually reinserted into the viral genome and studied in the viral context. The primary analysis showed the ability of certain mutants to complement the M94 deletion, thereby identifying the essential regions in the M94 protein. Secondly, the M94 mutants were re-inserted as second gene and analysed for their inhibitory capacity of the M94 wild type (wt) functions to identify dominant negative (DN) mutants. The inhibitory mutants found were then verified as DN mutants by regulated expression in the virus context and by analysis of their specific phenotype induced by overexpression. Further analysis of the DN mutant in comparison to a M94 deletion mutant revealed no effect of M94 in viral cleavage-packaging, despite previous publications. Additional analysis showed a block in secondary envelopment for both the DN mutant and the deletion mutant and the determination of the spread deficient phenotype of the M94 deletion virus. Altogether these results constitute a new model for secondary envelopment in β-Herpesvirinae.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 07/19
Human cytomegalovirus is a ubiquitous human pathogen, causing disease in the immunocompromised host. Most of its ORFs have not been well studied due to a limited host range and slow growth of HCMV in cultured cells. MCMV, a natural pathogen isolated from mice, constitutes the most amenable animal model for human β-herpesviruses. To date most of its approximately 200 genes have an unknown function. For the analysis of these genes straightforward mutagenesis methods are necessary. With the cloning of herpesviruses as an infectious bacterial artificial chromosome a novel approach of mutagenesis has been established. Herpesviruses are now accessible to the tools of bacterial genetics. Since then site-directed mutagenesis by homologous recombination using linear DNA fragments and random transposon BAC mutagenesis have been introduced to delineate the functions of viral ORFs. The purpose of this work was to analyze two members of the US 22 gene homolog family, genes m139 and m142, with site-directed mutagenesis. Members of this family are conserved in all herpesviruses and mostly have unknown functions. Transposon mutants showed a macrophage phenotype for m139, whereas m142 was possibly essential for viral replication. Genes m139-m141 and m142-m143 have complex transcriptional regions and have 3´-coterminal transcripts. The insertion of a 3-kb large transposon could destabilize the upstream transcripts. Site-directed mutants of genes m139 and m142, where only the start codon is deleted, should not influence transcript stability and permit confirmation of the results obtained with transposon mutagenesis. Targeted mutants of MCMV BAC were constructed for ORF m139 (ΔATG-m139) and m142 (ΔATG-m142, ΔATG-m142/FRT) by homologous recombination using linear DNA fragments. Mutant ΔATG-m139 showed attenuated growth in peritoneal macrophages. This mutant, with the first two ATGs deleted, expressed a truncated protein of 61 kDa. Gene m139 seems to act in cooperation with genes m140 and m141 on the protein level. The site-directed MCMV BAC mutant of ORF m142 on the other hand could not reconstitute viral progeny in eukaryotic cells. The ORF of m142 was inserted an ectopic position and viral progeny was reconstituted with this revertant. Thus, it was shown that gene m142 is essential for viral replication. Further analysis of nonessential and essential genes of cytomegalovirus will be needed to understand CMV viral pathology and to develop vaccines for herpesvirus infection and vectors for gene therapy.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 02/06
Herpesviruses cause highly prevalent infections associated with usually mild symptoms resulting in life long latency. However, they can provoke fatal disease in susceptible individuals such as immunocompromised patients either in the context of primary infection or after reactivation from latency. Despite of their emerging medical importance, herpesvirus infections can so far only be controlled by antiviral therapy targeting viral DNA replication, accompanied with side effects and occurrence of resistant strains. In this work, a novel platform for drug discovery was established that is based on a protein complementation assay (PCA), which can be used to study viral protein-protein interactions in a simple cell based assay. In a PCA two inactive fragments of a reporter enzyme are fused to two interacting proteins. Interaction of the proteins leads to proximity of the enzyme fragments, followed by reconstitution of the reporter enzyme activity. Members of the UL34 and UL31 families are conserved herpesvirus proteins. They interact with one another forming the nuclear egress complex (NEC), which is essential for the export of viral capsids from the cell nucleus. This crucial protein-protein interaction might serve as a potential drug target for anti-herpesvirus chemotherapy. In this work the mutual binding sites of the two proteins were localized and studied for their conservation. A PCA was established for M50 and M53 – the NEC proteins of the murine cytomegalovirus (MCMV) - by fusion of the N- and C-terminal part of the TEM-1 ß-lactamase of E. coli. The assay was validated and applied to representative members of the three herpesvirus subfamilies. Cross-complementation assays showed that partners derived from the same subfamily can replace each other in the PCA, however, homologues from different subfamilies can not. This cross-complementation reflects the in vivo situation: the human cytomegalovirus (HCMV), but not the HSV-1 or PrV homologues are able to rescue the M50 or M53 null phenotype in the viral context of MCMV. The lack of complementation between the subfamilies is due to their diverged binding sites, which are located in all cases within the first conserved region of the UL31 family proteins. The study of the binding site in UL34 family members revealed a bipartite binding motif. With the aim of a future high-throughput inhibitor screen an in vitro NEC-PCA was established using purified fusion proteins of HCMV.
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Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 03/19
Das zur Familie der Herpesviridae, Unterfamilie Gammaherpesvirinae, gehörende murine Herpesvirus 68 (MHV-68) besitzt mit 118 kbp wie die zu den Betaherpesvirinae gehörenden murinen und humanen Cytomegalieviren (MCMV und HCMV) mit ca. 230 kbp ein sehr großes Genom. Die Mehrzahl der 80 offenen Leserahmen wurde noch nicht charakterisiert. Zur Untersuchung dieser Gene ist die zufällige Herstellung viraler Mutanten die effiziente Methode, um zu neuen Erkenntnissen bezüglich ihrer Funktion zu gelangen. Aufgrund des großen Anteils nicht oder nur wenig charakterisierter Gene am viralen Genom und der oftmals nur geringen Homologie zu offenen Leserahmen anderer nah verwandter Herpesviren ist für MHV-68 ein Verfahren der „forward genetics“ diejenige Möglichkeit, welche den größten Erfolg verspricht. Dies beinhaltet eine ungezielte Mutagenese des viralen Genoms mit blinder Rekonstitution und nachfolgender Charakterisierung derjenigen Rekombinanten, welche einen interessanten Phänotypen zeigten. Bisher war die Herstellung rekombinanter MHV-68-Klone sehr arbeitsintensiv, da man diese gänzlich mit den Mitteln durchführen musste, welche die Methodik der Zellkultur zur Verfügung stellte. Aufgrund der Klonierung des MHV-68-Genoms als ein künstliches bakterielles Chromosom (BAC), der Etablierung des Verfahrens der Transposonmutagenese und mittels der Möglichkeit der Rekonstitution viraler Rekombinanten durch invasive Bakterien wurde diese Methode der klassischen oder forward genetics möglich. In dieser Arbeit wurde erstmals eine Mutagenese des Genoms von MHV-68 durchgeführt, die resultierenden viralen Rekombinanten rekonstituiert und einer phänotypischen Untersuchung unterzogen. Hierbei wurde das Wachstum der Mutanten auf sechs verschiedenen Zelllinien verglichen. Dies sollte grob augenscheinliche Unterschiede der rekombinanten Viren im Vergleich zu einer Wildtyp-Kontrolle nachweisen, wobei die von Brune et al. bereits etablierte Methodik an die Erfordernisse des MHV-68-Genoms angepasst werden konnte. Bezüglich des viralen Wachstums in Endothelzellen auffällig erscheinende Mutanten zeigten eine Häufung der Tn-Insertionen in der Genregion um ORF 10. Wachstumskurven bestätigten die Rolle von ORF 10 für die Fähigkeit des Virus in Endothelzellen zu replizieren. Dieses ist der erste Hinweis für eine interessante biologische Funktion dieses viralen ORFs, die in weiteren Arbeiten zu sichern und zu analysieren ist.
We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses.
In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) coliocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, BOhler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66,4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51 % (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the elose relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection.
The gene encoding glycoprotein B (gB) of murine cytomegalovirus (MCMV) strain Smith was identified, sequenced, and expressed by recombinant vaccinia virus. The gB gene was found adjacent to the polymerase gene, as it is in the genome of human cytomegalovirus (HCMV). The open reading frame consists of 2,784 nucleotides capable of encoding a protein of 928 amino acids. Comparison with gB homologs of other herpesviruses revealed a high degree of homology. The similarity between the MCMV gB and the HCMV gB is most prominent, since 45% of the amino acids are identical. In addition, all cysteine residues are at homologous positions, indicating a similar tertiary structure of the two proteins. In contrast to HCMV, the MCMV gB mRNA is a true late transcript. A recombinant vaccinia virus expressing the MCMV gB gene has been constructed (Vac-gB). Antibodies raised against the Vac-gB recombinant precipitated proteins of 130, 105, and 52 kDa from MCMV-infected cells. The identity of the MCMV gB with the major envelope glycoprotein of MCMV described by Loh et al. was shown (L. C. Loh, N. Balachandran, and L. F. Qualtiere, Virology 166:206-216, 1988). Immunization of mice with the Vac-gB recombinant gave rise to neutralizing antibodies.
A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and −524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.