H2B homology region of major immediate–early protein 1 is essential for murine cytomegalovirus to disrupt nuclear domain 10, but is not important for viral replication in cell culture

Cytomegalovirus (CMV) major immediate–early protein 1 (IE1) has multiple functions and is important for efficient viral infection. As does its counterpart in human CMV, murine CMV (MCMV) IE1 also functions as a disruptor of mouse-cell nuclear domain 10 (ND10), where many different gene-regulation proteins congregate. It still remains unclear how MCMV IE1 disperses ND10 and whether this dispersion could have any effect on viral replication. MCMV IE1 is 595 aa long and has multiple functional domains that have not yet been fully analysed. In this study, we dissected the IE1 molecule by truncation and/or deletion and found that the H2B homology domain (amino acid sequence NDIFERI) is required for the dispersion of ND10 by IE1. Furthermore, we made additional deletions and point mutations and found that the minimal truncation in the H2B homology domain required for IE1 to lose the ability to disperse ND10 is just 3 aa (IFE). Surprisingly, the mutated IE1 still interacted with PML and co-localized with ND10 but failed to disperse ND10. This suggests that binding to ND10 key protein is essential to, but not sufficient for, the dispersal of ND10, and that some other unknown mechanism must be involved in this biological procedure. Finally, we generated MCMV with IFE-deleted IE1 (MCMVdlIFE) and its revertant (MCMVIFERQ). Although MCMVdlIFE lost the ability to disperse ND10, plaque assays and viral gene production assays showed that the deletion of IFE did not increase viral replication in cell culture. We conclude that the dispersion of ND10 appears not to be important for MCMV replication in a mouse-cell culture.

Download Full-text

BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection

Viruses ◽

10.3390/v10090466 ◽

2018 ◽

Vol 10 (9) ◽

pp. 466 ◽

Cited By ~ 5

Author(s):

Francesco Martelli ◽

Zongsong Wu ◽

Serena Delbue ◽

Fabian Weissbach ◽

Maria Giulioli ◽

...

Keyword(s):

Viral Replication ◽

Point Mutations ◽

T Antigen ◽

Host Cells ◽

Replication Capacity ◽

Viral Gene ◽

New Host ◽

Infected Cells ◽

Multiple Sclerosis Patients ◽

Coding Control

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

Download Full-text

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2214 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2214-2223 ◽

Cited By ~ 8

Author(s):

K T Jeang ◽

M S Cho ◽

G S Hayward

Keyword(s):

Cell Line ◽

Mammalian Cells ◽

Regulatory Region ◽

Constitutive Expression ◽

Regulatory Elements ◽

Mouse Cell ◽

Cellular Protein ◽

Immediate Early ◽

Early Protein ◽

Infected Cells

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

Download Full-text

Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

Viruses ◽

10.3390/v6020808 ◽

2014 ◽

Vol 6 (2) ◽

pp. 808-831 ◽

Cited By ~ 5

Author(s):

Annette Fink ◽

Julia Büttner ◽

Doris Thomas ◽

Rafaela Holtappels ◽

Matthias Reddehase ◽

...

Keyword(s):

T Cell ◽

Cell Epitope ◽

Cd8 T Cell ◽

Immediate Early ◽

Murine Cytomegalovirus ◽

T Cell Epitope ◽

Upstream Gene ◽

Early Protein

Download Full-text

The Immediate-Early Protein IE0 of the Autographa californica Nucleopolyhedrovirus Is Not Essential for Viral Replication

Journal of Virology ◽

10.1128/jvi.79.18.12122.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 12122-12122 ◽

Cited By ~ 4

Author(s):

Liqun Lu ◽

Hadassah Rivkin ◽

Nor Chejanovsky

Keyword(s):

Viral Replication ◽

Autographa Californica ◽

Immediate Early ◽

Early Protein ◽

Autographa Californica Nucleopolyhedrovirus

Download Full-text

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

Virology ◽

10.1016/j.virol.2016.02.012 ◽

2016 ◽

Vol 492 ◽

pp. 82-91 ◽

Cited By ~ 4

Author(s):

Mohamed I. Khalil ◽

Xibing Che ◽

Phillip Sung ◽

Marvin H. Sommer ◽

John Hay ◽

...

Keyword(s):

Viral Replication ◽

Varicella Zoster Virus ◽

Mutational Analysis ◽

Immediate Early ◽

Varicella Zoster ◽

Early Protein

Download Full-text

Suppression of pseudorabies virus replication by a mutant form of immediate-early protein IE180 repressing the viral gene transcription

Veterinary Microbiology ◽

10.1016/s0378-1135(97)00153-3 ◽

1998 ◽

Vol 60 (2-4) ◽

pp. 107-117 ◽

Cited By ~ 4

Author(s):

E Ono

Keyword(s):

Virus Replication ◽

Gene Transcription ◽

Pseudorabies Virus ◽

Immediate Early ◽

Viral Gene ◽

Mutant Form ◽

Viral Gene Transcription ◽

Early Protein

Download Full-text

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription.

Journal of Virology ◽

10.1128/jvi.58.1.59-66.1986 ◽

1986 ◽

Vol 58 (1) ◽

pp. 59-66 ◽

Cited By ~ 30

Author(s):

U H Koszinowski ◽

G M Keil ◽

H Volkmer ◽

M R Fibi ◽

A Ebeling-Keil ◽

...

Keyword(s):

Gene Transcription ◽

Immediate Early ◽

Murine Cytomegalovirus ◽

Early Protein

Download Full-text

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

Journal of Virology ◽

10.1128/jvi.76.1.313-326.2002 ◽

2002 ◽

Vol 76 (1) ◽

pp. 313-326 ◽

Cited By ~ 49

Author(s):

Jeffery L. Meier ◽

Michael J. Keller ◽

James J. McCoy

Keyword(s):

Gene Expression ◽

Viral Replication ◽

Gene Transcription ◽

Human Cytomegalovirus ◽

Viral Gene Expression ◽

Immediate Early ◽

Viral Gene ◽

Distal Enhancer ◽

Cis Acting ◽

Hcmv Replication

ABSTRACT We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Download Full-text