scholarly journals Actin-based motility drives baculovirus transit to the nucleus and cell surface

2010 ◽  
Vol 190 (2) ◽  
pp. 187-195 ◽  
Author(s):  
Taro Ohkawa ◽  
Loy E. Volkman ◽  
Matthew D. Welch
Keyword(s):  
Gene Expression ◽  
Actin Polymerization ◽  
Nuclear Periphery ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Evolutionary Strategy ◽  
Viral Gene ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Entry

Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells

2009 ◽  
Vol 83 (10) ◽  
pp. 4895-4911 ◽  
Author(s):  
Hari Raghu ◽  
Neelam Sharma-Walia ◽  
Mohanan Valiya Veettil ◽  
Sathish Sadagopan ◽  
Bala Chandran
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Actin Polymerization ◽  
Umbilical Vein ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Virus Particles ◽  
Huvec Cells ◽  
D Cells ◽  
Infectious Entry

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes clathrin-mediated endocytosis for its infectious entry into human foreskin fibroblast (HFF) cells (S. M. Akula, P. P. Naranatt, N.-S. Walia, F.-Z. Wang, B. Fegley, and B. Chandran, J. Virol. 77:7978-7990, 2003). Here, we characterized KSHV entry into primary human microvascular dermal endothelial (HMVEC-d) and human umbilical vein endothelial (HUVEC) cells. Similar to the results for HMVEC-d cells, KSHV infection of HUVEC cells also resulted in an initial high level and subsequent decline in the expression of the lytic switch gene, ORF50, while latent gene expression persisted. Internalized virus particles enclosed in irregular vesicles were observed by electron microscopy of infected HMVEC-d cells. At an early time of infection, colocalization of KSHV capsid with envelope was observed by immunofluorescence analysis, thus demonstrating endocytosis of intact enveloped virus particles. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and filipin (C35H58O11), a caveolar endocytosis inhibitor, did not have any effect on KSHV binding, entry (DNA internalization), or gene expression in HMVEC-d and HUVEC cells. In contrast to the results for HFF cells, virus entry and gene expression in both types of endothelial cells were significantly blocked by macropinocytosis inhibitors (EIPA [5-N-ethyl-N-isoproamiloride] and rottlerin [C30H28O8]) and by cytochalasin D, which affects actin polymerization. Inhibition of lipid raft blocked viral gene expression in HMVEC-d cells but not in HUVEC or HFF cells. In HMVEC-d and HUVEC cells, KSHV induced the actin polymerization and formation of lamellipodial extensions that are essential for macropinocytosis. Inhibition of macropinocytosis resulted in the distribution of viral capsids at the HMVEC-d cell periphery, and capsids did not associate with microtubules involved in the nuclear delivery of viral DNA. Internalized KSHV in HMVEC-d and HUVEC cells colocalized with the macropinocytosis marker dextran and not with the clathrin pathway marker transferrin or with caveolin. Dynasore, an inhibitor of dynamin, did not block viral entry into endothelial cells but did inhibit entry into HFF cells. KSHV was not associated with the early endosome marker EEA-1 in HMVEC-d cells, but rather with the late endosome marker LAMP1, as well as with Rab34 GTPase that is known to regulate macropinocytosis. Silencing Rab34 with small interfering RNA dramatically inhibited KSHV gene expression. Bafilomycin-mediated disruption of endosomal acidification inhibited viral gene expression. Taken together, these findings suggest that KSHV utilizes the actin polymerization-dependent, dynamin-independent macropinocytic pathway that involves a Rab34 GTPase-dependent late endosome and low-pH environment for its infectious entry into HMVEC-d and HUVEC cells. These studies also demonstrate that KSHV utilizes different modes of endocytic entry in fibroblast and endothelial cells.

scholarly journals Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

2014 ◽  
Vol 88 (22) ◽  
pp. 13249-13259 ◽  
Author(s):  
Y. Lu ◽  
T. J. Kucharski ◽  
I. Gamache ◽  
P. Blanchette ◽  
P. E. Branton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Gene Expression ◽  
Adenovirus Type ◽  
Viral Gene ◽  
Nuclear Pore ◽  
Adenovirus Type 5

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 CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus 18 Genome Regulates Viral Oncogene Expression

2015 ◽  
Vol 89 (9) ◽  
pp. 4770-4785 ◽  
Author(s):  
Christian Paris ◽  
Ieisha Pentland ◽  
Ian Groves ◽  
David C. Roberts ◽  
Simon J. Powis ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Risk ◽  
Host Cell ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Viral Gene ◽  
Genome Wide ◽  
High Risk Hpv

ABSTRACTHost cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.IMPORTANCEThe establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

scholarly journals Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle

2020 ◽  
Vol 134 (5) ◽  
pp. jcs252544
Author(s):  
Maarit Suomalainen ◽  
Vibhu Prasad ◽  
Abhilash Kannan ◽  
Urs F. Greber
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Molecule ◽  
Click Reaction ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Viral Gene ◽  
Viral Genomes ◽  
E1a Gene ◽  
G1 Cells

ABSTRACTIn clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2′-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide–alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

Peripheral Localization and Regulation of the Murine β-Globin Locus.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4070-4070
Author(s):  
Tobias Ragoczy ◽  
Agnes Telling ◽  
Rachel Byron ◽  
M.A. Bender ◽  
Mark Groudine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Nuclear Periphery ◽  
Embryonic Stem ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Erythroid Progenitor ◽  
Interphase Cell ◽  
Pore Complexes ◽  
Globin Gene Expression

Abstract The interphase cell nucleus is structurally and functionally compartmentalized, making the subnuclear position of genes an important determinant of their activity. During cellular differentiation, as tissue-specific expression programs unfold, dynamic reorganization of the nucleus positions sets of genes in active or repressive compartments. The nuclear periphery has emerged as an unusually complex compartment in this process. While it is marked by facultative heterochromatin and has been considered primarily as a repressive compartment, recent work suggests that active genes may also associate with the periphery. Moreover, the nuclear envelope is riddled with nuclear pore complexes, the gateways for molecular exchange between the cytoplasm and the nucleus, resulting in substantial traffic through this compartment. Using murine erythropoiesis as a model system, our recent three dimensional analysis of the positioning of the β-globin locus revealed that, when inactive in undifferentiated embryonic stem cells and erythroid progenitor cells, the locus is positioned predominantly at the nuclear periphery and appears to contact the lamina. This association is lost with progressing erythroid maturation, and the locus is repositioned towards the nuclear interior concomitant with increasing β-globin gene expression. Importantly, however, β-major globin expression begins at the nuclear periphery prior to relocalization, suggesting that associations of the locus at the periphery may shift from repressive to activating complexes. We are investigating the interactions of the β-globin locus with the nuclear periphery by two approaches: Using enhanced imaging permitted by Cryo-ImmunoFISH, we are analyzing the position of the locus relative to specific components of the nuclear lamina and nuclear pore complexes at distinct differentiation stages. In addition, we are probing the physical interactions of the locus with the periphery by biochemical means. To this end we are using ChIP-chip to identify lamina associated proteins binding the β-globin locus and to determine what sequence elements within the locus mediate these interactions. Ultimately these experiments will shed further light on mechanisms regulating β-globin gene expression during erythropoiesis and how stage-specific nuclear localization contributes to this process.

scholarly journals Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Thomas M. Kristie
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Early Gene ◽  
Productive Infection ◽  
Viral Gene ◽  
Early Protein ◽  
Protein Icp0 ◽  
Simplex Virus

ABSTRACT Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes.

scholarly journals Cell Cycle-Independent Expression of Immediate-Early Gene 3 Results in G1 and G2 Arrest in Murine Cytomegalovirus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10188-10198 ◽  
Author(s):  
Lüder Wiebusch ◽  
Anke Neuwirth ◽  
Linus Grabenhenrich ◽  
Sebastian Voigt ◽  
Christian Hagemeier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Viral Gene Expression ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Viral Gene ◽  
Cycle Arrest ◽  
Infected Cells

ABSTRACT The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G0/G1 phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G1 DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G1. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G2. Moreover, MCMV can also replicate in G2 cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G1 or G2. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.

scholarly journals Small Internal Deletions in the Human Cytomegalovirus IE2 Gene Result in Nonviable Recombinant Viruses with Differential Defects in Viral Gene Expression

2004 ◽  
Vol 78 (4) ◽  
pp. 1817-1830 ◽  
Author(s):  
Elizabeth A. White ◽  
Charles L. Clark ◽  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Early Gene ◽  
Immediate Early ◽  
Viral Gene ◽  
Recombinant Viruses ◽  
Late Genes

ABSTRACT The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86Δ356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86Δ427-435 and IE2 86Δ505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.

scholarly journals HAdV protein V core protein is targeted by the host SUMOylation machinery to limit essential viral functions

2017 ◽  
pp. JVI.01451-17 ◽  
Author(s):  
Nora Freudenberger ◽  
Tina Meyer ◽  
Peter Groitl ◽  
Thomas Dobner ◽  
Sabrina Schreiner
Keyword(s):  
Gene Expression ◽  
Core Protein ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Virus Growth ◽  
Core Proteins ◽  
Consensus Motifs

Human Adenoviruses (HAdV) are non-enveloped containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear-pore-complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood.The core protein V is speculated to bridge core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultanously, these altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than observed during wildtype infection.Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies.ImportanceMany decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion, as the functions are more diverse. We showed that capsid protein protein VI is regulating antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, being beneficial for efficient viral gene expression. Here, we were able to show that also core protein V represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. Conclusively, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency.

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