Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34<sup>+</sup>Cell Populations

Albright, Emily R.; Kalejta, Robert F.

doi:10.1128/jvi.01436-13

Cited by 43 publications

(54 citation statements)

References 67 publications

(128 reference statements)

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“…Cells were lysed in either radioimmunoprecipitation assay (RIPA; Thermo), 1% SDS, or 1ϫ passive lysis (Promega) buffer, and equal amounts of lysates were separated by SDS-PAGE, transferred to Optitran (GE Healthcare) or polyvinylidene difluoride (PVDF; Millipore) membranes, and analyzed by Western blotting as previously described (9,30). For indirect immunofluorescence, adherent NHDFs and suspension THP-1 or CD34 ϩ cells were washed with phosphate-buffered saline (PBS), fixed in 1% paraformaldehyde, and processed as previously described (20,21,29).…”

Section: Methodsmentioning

confidence: 99%

“…MIEP-directed transcription initiates productive replication in fibroblasts when the viral pp71 protein, a component of the virion tegument delivered upon infection, migrates to the nucleus and degrades Daxx, thereby counteracting histone deacetylase (HDAC)-mediated repression (29). When HCMV infects incompletely differentiated myeloid cells, in which it enters latency, tegument-delivered pp71 remains in the cytoplasm and IE1 transcription is suppressed (20,21,30,31). For high-passagenumber laboratory strains, the intrinsic defense instituted by Daxx may be the only significant restriction to IE1 transcription during latency because IE1 transcription from AD169 can be rescued in THP-1 and CD34 ϩ cells (20,21) by either Daxx knockdown or the HDAC inhibitors trichostatin A (TSA) or valproic acid (VPA).…”

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confidence: 99%

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Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Lee

Caviness

Albright

et al. 2016

J Virol

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The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34؉ hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo-and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. IMPORTANCELatency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency but may do so in temporally distinct manners. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs. Human cytomegalovirus (HCMV) is a betaherpesvirus that causes birth defects and disease in immunocompromised and immunosuppressed patients and has been associated with cancers, cardiovascular disease, and immune dysfunction (1-3). HCMV productively (lytically) infects differentiated cells, such as fibroblasts, endothelial cells, and epithelial cells, and latently infects incompletely differentiated cells of the myeloid lineage, such as monocytes and CD34 ϩ hematopoietic progenitor cells (HPC) (4, 5). Productive infection in multiple cell types within the human host facilitates viral dissemination, while latency ensures lifelong persistence despite an effective immune response against lyticphase antigens. Periodic reactivations of latent infections into the productive phase perpetuate both lifelong infecti...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Lee

Caviness

Albright

et al. 2016

J Virol

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“…Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors or 1% SDS containing 2% ␤-mercaptoethanol, and equal amounts of lysates were separated by SDS-PAGE, transferred to Optitran membranes (GE Healthcare), and analyzed by Western blotting as previously described (60).…”

Section: Cells and Infectionsmentioning

confidence: 99%

Canonical and Variant Forms of Histone H3 Are Deposited onto the Human Cytomegalovirus Genome during Lytic and Latent Infections

Albright

Kalejta

2016

J Virol

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Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes and which variant forms of the core histone proteins are deposited are incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histones H3.1 and H3.2 (collectively, H3.1/2) and H3.3 during lytic and latent HCMV infections. Here, we show that they are deposited on HCMV genomes during lytic and latent infections, suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating that both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections. IMPORTANCEIt is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways and viral fate determinations (lytic or latent) and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how the genomes are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing that HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to posttranslational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.T he large genomes of eukaryotic cells must be highly compacted in order to fit within the restricted volume of the nucleus. To accomplish this, eukaryotic DNA is packaged into a repeating nucleoprotein structure known as chromatin, the basic subunit of which is the nucleosome (1, 2). The core nucleosome particle consists of approximately 146 bp of DNA wrapped nearly twice around a histone hetero-octamer consisting of two copies each of the four core histone proteins H2A, H2B, H3, and H4 (3). Nucleosomes are further compacted into higher-order chromatin fibers through the incorporation of the linker histone H1, other nonhistone proteins, and structural RNA components (4-6).Wrapping DNA in nucleoso...

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“…21,30,[35][36][37][45][46][47] However, it is clear that some experimental models using established cell lines do not appear to fully recapitulate all aspects of control of latency and reactivation observed in primary myeloid cells. 48 A totally quiescent viral genome during latent infection would clearly be the ideal way to avoid immune surveillance-if viral proteins are not expressed at all there would be no processing and presentation of viral antigens to specific T cells and, thus, latently infected cells would be ignored by the host immune response. However, recent work has shown that the viral gene expression is far from quiescent during latent infection and that expression of a number of viral transcripts, encoding viral proteins, routinely occurs.…”

Section: Establishment Of Latency and The Molecular Biology Of The Lamentioning

confidence: 99%

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

et al. 2014

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While the host immune response following primary human cytomegalovirus (HCMV) infection is generally effective at stopping virus replication and dissemination, virus is never cleared by the host and like all herpesviruses, persists for life. At least in part, this persistence is known to be facilitated by the ability of HCMV to establish latency in myeloid cells in which infection is essentially silent with, importantly, a total lack of new virus production. However, although the viral transcription programme during latency is much suppressed, a number of viral genes are expressed during latent infection at the protein level and many of these have been shown to have profound effects on the latent cell and its environment. Intriguingly, many of these latency-associated genes are also expressed during lytic infection. Therefore, why the same potent host immune responses generated during lytic infection to these viral gene products are not recognized during latency, thereby allowing clearance of latently infected cells, is far from clear. Reactivation from latency is also a major cause of HCMV-mediated disease, particularly in the immune compromised and immune naive, and is also likely to be a major source of virus in chronic subclinical HCMV infection which has been suggested to be associated with long-term diseases such as atherosclerosis and some neoplasias. Consequently, understanding latency and why latently infected cells appear to be immunoprivileged is crucial for an understanding of the pathogenesis of HCMV and may help to design strategies to eliminate latent virus reservoirs, at least in certain clinical settings.

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Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Cited by 43 publications

References 67 publications

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Canonical and Variant Forms of Histone H3 Are Deposited onto the Human Cytomegalovirus Genome during Lytic and Latent Infections

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

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Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+Cell Populations

Cited by 43 publications

References 67 publications

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Canonical and Variant Forms of Histone H3 Are Deposited onto the Human Cytomegalovirus Genome during Lytic and Latent Infections

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

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Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations