Long-Term Infection and Shedding of Human Cytomegalovirus in T98G Glioblastoma Cells

ABSTRACT Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, affecting primarily the central nervous system (CNS). To further understand this CNS pathology, cells from glioblastoma cell lines T98G and A172, the astrocytic glioblastoma cell line CCF-STTG1 (CCF), and the neuroblastoma cell line SH-SY5Y (SY5Y) were infected with HCMV. CCF and SY5Y cells were fully permissive for infection, while A172 cells were nonpermissive. In T98G cells, the majority of cells showed viral deposition into the nucleus by 6 h postinfection (hpi); however, viral immediate-early gene expression was observed in only ∼30% of cells in the first 72 h. In viral antigen (Ag)-positive cells, although the development of complete viral replication centers was delayed, fully developed centers formed by 96 hpi. Interestingly, even at very late times postinfection, a mixture of multiple small, bipolar, and large foci was always present. The initial trafficking of input pp65 into the nucleus was also delayed. Titer and infectious-center assays showed a small number of T98G cells shedding virus at very low levels. Surprisingly, both Ag-positive and Ag-negative cells continued to divide; because of this continuous division, we adopted a protocol for passaging the T98G cells every third day to prevent overcrowding. Under this protocol, detectable infectious-virus shedding continued until passage 5 and viral gene expression continued through eight passages. This evidence points to T98G cells as a promising model for long-term infections.

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

Journal of Virology ◽

10.1128/jvi.78.4.1817-1830.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1817-1830 ◽

Cited By ~ 63

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.

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Human cytomegalovirus immediate-early gene expression is restricted by the nuclear domain 10 component Sp100

Journal of General Virology ◽

10.1099/vir.0.030981-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1532-1538 ◽

Cited By ~ 44

Author(s):

Martina Adler ◽

Nina Tavalai ◽

Regina Müller ◽

Thomas Stamminger

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Viral Gene Expression ◽

Early Gene ◽

Immediate Early ◽

Viral Gene ◽

Major Protein ◽

Hcmv Replication ◽

Protein Components ◽

Nuclear Domains

Nuclear domains 10 (ND10s) are discrete subnuclear structures that contain the three major protein components promyelocytic leukaemia protein (PML), hDaxx and Sp100. Previous studies identified the ND10-components PML and hDaxx as cellular restriction factors that independently counteract human cytomegalovirus (HCMV) infection via the repression of viral immediate-early (IE) gene expression. Consequently, we asked whether Sp100 is likewise involved in this repressive activity. Infection of Sp100 knockdown (kd) cells with HCMV resulted in a significantly increased plaque-forming ability. In addition, ablation of Sp100 led to a considerable increase in the number of IE1-expressing cells, indicating that Sp100 suppresses the initiation of viral gene expression. Next, double-kd cells, lacking either Sp100/hDaxx or Sp100/PML, were generated. Here, infection resulted in an additional enhancement in HCMV replication efficacy compared with the single-kd cells. Thus, our results further strengthen the concept that the three major ND10-components independently contribute to the cellular restriction of HCMV replication.

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Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

Journal of General Virology ◽

10.1099/vir.0.038083-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 1046-1058 ◽

Cited By ~ 11

Author(s):

James C. Towler ◽

Bahram Ebrahimi ◽

Brian Lane ◽

Andrew J. Davison ◽

Derrick J. Dargan

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Growth Kinetics ◽

Viral Gene Expression ◽

Cell Types ◽

Viral Gene ◽

Genome Replication ◽

Cell Type ◽

Low Passage ◽

Temporal Expression Pattern

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

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HCMV Infection and Apoptosis: How Do Monocytes Survive HCMV Infection?

Viruses ◽

10.3390/v10100533 ◽

2018 ◽

Vol 10 (10) ◽

pp. 533 ◽

Cited By ~ 10

Author(s):

Donna Collins-McMillen ◽

Liudmila Chesnokova ◽

Byeong-Jae Lee ◽

Heather Fulkerson ◽

Reynell Brooks ◽

...

Keyword(s):

Gene Expression ◽

Cell Survival ◽

Hcmv Infection ◽

Viral Gene Expression ◽

Multiple Organ ◽

Viral Gene ◽

Blood Monocytes ◽

Induced Differentiation ◽

Organ Systems

Human cytomegalovirus (HCMV) infection of peripheral blood monocytes plays a key role in the hematogenous dissemination of the virus to multiple organ systems following primary infection or reactivation of latent virus in the bone marrow. Monocytes have a short life span of 1–3 days in circulation; thus, HCMV must alter their survival and differentiation to utilize these cells and their differentiated counterparts—macrophages—for dissemination and long term viral persistence. Because monocytes are not initially permissive for viral gene expression and replication, HCMV must control host-derived factors early during infection to prevent apoptosis or programmed cell death prior to viral induced differentiation into naturally long-lived macrophages. This review provides a short overview of HCMV infection of monocytes and describes how HCMV has evolved to utilize host cell anti-apoptotic pathways to allow infected monocytes to bridge the 48–72 h viability gate so that differentiation into a long term stable mature cell can occur. Because viral gene expression is delayed in monocytes following initial infection and only occurs (begins around two to three weeks post infection in our model) following what appears to be complete differentiation into mature macrophages or dendritic cells, or both; virally-encoded anti-apoptotic gene products cannot initially control long term infected cell survival. Anti-apoptotic viral genes are discussed in the second section of this review and we argue they would play an important role in long term macrophage or dendritic cell survival following infection-induced differentiation.

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Differential viral gene expression and its effect on the biological properties of the cell clones of an HIV-1-infected cell line

Virology ◽

10.1016/0042-6822(90)90495-d ◽

1990 ◽

Vol 177 (1) ◽

pp. 380-383 ◽

Cited By ~ 4

Author(s):

V.S. Kalyanaraman ◽

V. Rodriguez ◽

S. Josephs ◽

R.C. Gallo ◽

M.G. Sarngadharan

Keyword(s):

Gene Expression ◽

Infected Cell ◽

Cell Line ◽

Viral Gene Expression ◽

Biological Properties ◽

Viral Gene ◽

Infected Cell Line ◽

Cell Clones ◽

Hiv 1

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

The Journal of Cell Biology ◽

10.1083/jcb.201001162 ◽

2010 ◽

Vol 190 (2) ◽

pp. 187-195 ◽

Cited By ~ 143

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.

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Global Analysis of Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Gene Expression in the Midgut of the Lepidopteran HostTrichoplusia ni

Journal of Virology ◽

10.1128/jvi.01277-18 ◽

2018 ◽

Vol 92 (23) ◽

Cited By ~ 12

Author(s):

Anita Shrestha ◽

Kan Bao ◽

Yun-Ru Chen ◽

Wenbo Chen ◽

Ping Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Trichoplusia Ni ◽

Cultured Cells ◽

Expression Profiles ◽

Secondary Infection ◽

Expression Patterns ◽

Viral Gene Expression ◽

Viral Gene ◽

Viral Genes

ABSTRACTThe baculovirusAutographa californica multiple nucleopolyhedrovirus(AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes inTrichoplusia nimidgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from aT. nicell line (Tnms42). Several viral genes (p6.9,orf76,orf75,pp31,Ac-bro,odv-e25, andodv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polhandp10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.IMPORTANCEBaculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut inTrichoplusia niand compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.

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Repression of human cytomegalovirus major immediate early gene expression in a monocytic cell line

Journal of General Virology ◽

10.1099/0022-1317-73-2-433 ◽

1992 ◽

Vol 73 (2) ◽

pp. 433-435 ◽

Cited By ~ 60

Author(s):

J. H. Sinclair ◽

J. Baillie ◽

L. A. Bryant ◽

J. A. Taylor-Wiedeman ◽

J. G. P. Sissons

Keyword(s):

Gene Expression ◽

Cell Line ◽

Human Cytomegalovirus ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Early Gene Expression

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Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic ‘pre-immediate-early’ repression of viral gene expression mediated by histone post-translational modification

Journal of General Virology ◽

10.1099/vir.0.012526-0 ◽

2009 ◽

Vol 90 (10) ◽

pp. 2364-2374 ◽

Cited By ~ 56

Author(s):

Ian J. Groves ◽

Matthew B. Reeves ◽

John H. Sinclair

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Chromatin Structure ◽

Viral Gene Expression ◽

Lytic Infection ◽

Immediate Early ◽

Late Gene ◽

Viral Gene ◽

Gene Promoters ◽

Late Gene Expression

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at ‘pre-IE’ times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

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