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Open AccessFeature PaperArticle

Modeling HSV-1 Latency in Human Embryonic Stem Cell-Derived Neurons

by Aldo Pourchet, Aram S. Modrek, Dimitris G. Placantonakis, Ian Mohr and Angus C. Wilson

Pathogens 2017, 6(2), 24; https://doi.org/10.3390/pathogens6020024 - 08 Jun 2017

Cited by 38 | Viewed by 6639

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems [...] Read more.

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-α. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools. Full article

(This article belongs to the Special Issue Herpesviruses)

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13085 KiB

Open AccessFeature PaperArticle

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

by A. Gregory Bruce, Serge Barcy, Terri DiMaio, Emilia Gan, H. Jacques Garrigues, Michael Lagunoff and Timothy M. Rose

Pathogens 2017, 6(1), 11; https://doi.org/10.3390/pathogens6010011 - 19 Mar 2017

Cited by 30 | Viewed by 6282

Abstract

The transcriptome of the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial [...] Read more.

The transcriptome of the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial induction, and a comprehensive annotation of the KSHV genome was determined. A set of unique coding sequence (UCDS) features and a process to resolve overlapping transcripts were developed to accurately quantitate transcript levels from specific promoters. Similar patterns of KSHV expression were detected in BCBL-1 cells undergoing long-term latent infections and in primary latent infections of both BEC and LEC cultures. High expression levels of poly-adenylated nuclear (PAN) RNA and spliced and unspliced transcripts encoding the K12 Kaposin B/C complex and associated microRNA region were detected, with an elevated expression of a large set of lytic genes in all latently infected cultures. Quantitation of non-overlapping regions of transcripts across the complete KSHV genome enabled for the first time accurate evaluation of the KSHV transcriptome associated with viral latency in different cell types. Hierarchical clustering applied to a gene correlation matrix identified modules of co-regulated genes with similar correlation profiles, which corresponded with biological and functional similarities of the encoded gene products. Gene modules were differentially upregulated during latency in specific cell types indicating a role for cellular factors associated with differentiated and/or proliferative states of the host cell to influence viral gene expression. Full article

(This article belongs to the Special Issue Herpesviruses)

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Open AccessArticle

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

by Alexis L. Santana, Darby G. Oldenburg, Varvara Kirillov, Laraib Malik, Qiwen Dong, Roman Sinayev, Kenneth B. Marcu, Douglas W. White and Laurie T. Krug

Pathogens 2017, 6(1), 9; https://doi.org/10.3390/pathogens6010009 - 16 Feb 2017

Cited by 9 | Viewed by 6926

Abstract

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host [...] Read more.

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA. Full article

(This article belongs to the Special Issue Herpesviruses)

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Open AccessArticle

Neurotrophic Factors NGF, GDNF and NTN Selectively Modulate HSV1 and HSV2 Lytic Infection and Reactivation in Primary Adult Sensory and Autonomic Neurons

by Andy A. Yanez, Telvin Harrell, Heather J. Sriranganathan, Angela M. Ives and Andrea S. Bertke

Pathogens 2017, 6(1), 5; https://doi.org/10.3390/pathogens6010005 - 07 Feb 2017

Cited by 20 | Viewed by 5814

Abstract

Herpes simplex viruses (HSV1 and HSV2) establish latency in peripheral ganglia after ocular or genital infection, and can reactivate to produce different patterns and frequencies of recurrent disease. Previous studies showed that nerve growth factor (NGF) maintains HSV1 latency in embryonic sympathetic and [...] Read more.

Herpes simplex viruses (HSV1 and HSV2) establish latency in peripheral ganglia after ocular or genital infection, and can reactivate to produce different patterns and frequencies of recurrent disease. Previous studies showed that nerve growth factor (NGF) maintains HSV1 latency in embryonic sympathetic and sensory neurons. However, adult sensory neurons are no longer dependent on NGF for survival, some populations cease expression of NGF receptors postnatally, and the viruses preferentially establish latency in different populations of sensory neurons responsive to other neurotrophic factors (NTFs). Thus, NGF may not maintain latency in adult sensory neurons. To identify NTFs important for maintaining HSV1 and HSV2 latency in adult neurons, we investigated acute and latently-infected primary adult sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) after NTF removal. NGF and glial cell line-derived neurotrophic factor (GDNF) deprivation induced HSV1 reactivation in adult sympathetic neurons. In adult sensory neurons, however, neurturin (NTN) and GDNF deprivation induced HSV1 and HSV2 reactivation, respectively, while NGF deprivation had no effects. Furthermore, HSV1 and HSV2 preferentially reactivated from neurons expressing GFRα2 and GFRα1, the high affinity receptors for NTN and GDNF, respectively. Thus, NTN and GDNF play a critical role in selective maintenance of HSV1 and HSV2 latency in primary adult sensory neurons. Full article

(This article belongs to the Special Issue Herpesviruses)

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Review

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Open AccessFeature PaperReview

KSHV and the Role of Notch Receptor Dysregulation in Disease Progression

by Jennifer L. DeCotiis and David M. Lukac

Pathogens 2017, 6(3), 34; https://doi.org/10.3390/pathogens6030034 - 04 Aug 2017

Cited by 12 | Viewed by 5793

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi’s Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman’s Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its [...] Read more.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi’s Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman’s Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis. Full article

(This article belongs to the Special Issue Herpesviruses)

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Open AccessReview

Herpes Simplex Virus Establishment, Maintenance, and Reactivation: In Vitro Modeling of Latency

by Nikki M. Thellman and Steven J. Triezenberg

Pathogens 2017, 6(3), 28; https://doi.org/10.3390/pathogens6030028 - 23 Jun 2017

Cited by 41 | Viewed by 11811

Abstract

All herpes viruses establish lifelong infections (latency) in their host, and herpes simplex viruses (HSVs) are highly prevalent worldwide. Recurrence of HSV infections contributes to significant disease burden in people and on rare occasion can be fatal. Cell culture models that recapitulate latent [...] Read more.

All herpes viruses establish lifelong infections (latency) in their host, and herpes simplex viruses (HSVs) are highly prevalent worldwide. Recurrence of HSV infections contributes to significant disease burden in people and on rare occasion can be fatal. Cell culture models that recapitulate latent infection provide valuable insight on the host processes regulating viral establishment and maintenance of latency. More robust and rapid than infections in live animal studies, advancements in neuronal culture techniques have made the systematic analysis of viral reactivation mechanisms feasible. Only recently have human neuronal cell lines been available, but models in the natural host cell are a critical addition to the currently available models. Full article

(This article belongs to the Special Issue Herpesviruses)

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Open AccessReview

Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease

by Marshall V. Williams, Brandon Cox and Maria Eugenia Ariza

Pathogens 2017, 6(1), 2; https://doi.org/10.3390/pathogens6010002 - 28 Dec 2016

Cited by 21 | Viewed by 9169

Abstract

The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person’s lifetime. While it is well accepted that human herpesviruses are implicated in [...] Read more.

The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person’s lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein–Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer. Full article

(This article belongs to the Special Issue Herpesviruses)

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Open AccessReview

Varicella-Zoster Virus Infectious Cycle: ER Stress, Autophagic Flux, and Amphisome-Mediated Trafficking

by Charles Grose, Erin M. Buckingham, John E. Carpenter and Jeremy P. Kunkel

Pathogens 2016, 5(4), 67; https://doi.org/10.3390/pathogens5040067 - 10 Dec 2016

Cited by 19 | Viewed by 8949

Abstract

Varicella-zoster virus (VZV) induces abundant autophagy. Of the nine human herpesviruses, the VZV genome is the smallest (~124 kbp), lacking any known inhibitors of autophagy, such as the herpes simplex virus ICP34.5 neurovirulence gene. Therefore, this review assesses the evidence for VZV-induced cellular [...] Read more.

Varicella-zoster virus (VZV) induces abundant autophagy. Of the nine human herpesviruses, the VZV genome is the smallest (~124 kbp), lacking any known inhibitors of autophagy, such as the herpes simplex virus ICP34.5 neurovirulence gene. Therefore, this review assesses the evidence for VZV-induced cellular stress, endoplasmic-reticulum-associated degradation (ERAD), and autophagic flux during the VZV infectious cycle. Even though VZV is difficult to propagate in cell culture, the biosynthesis of the both N- and O-linked viral glycoproteins was found to be abundant. In turn, this biosynthesis provided evidence of endoplasmic reticulum (ER) stress, including a greatly enlarged ER and a greatly diminished production of cellular glycoproteins. Other signs of ER stress following VZV infection included detection of the alternatively spliced higher-molecular-weight form of XBP1 as well as CHOP. VZV infection in cultured cells leads to abundant autophagosome production, as was visualized by the detection of the microtubule-associated protein 1 light chain 3-II (LC3-II). The degree of autophagy induced by VZV infection is comparable to that induced in uninfected cells by serum starvation. The inhibition of autophagic flux by chemicals such as 3-methyladenine or ATG5 siRNA, followed by diminished virus spread and titers, has been observed. Since the latter observation pointed to the virus assembly/trafficking compartments, we purified VZ virions by ultracentrifugation and examined the virion fraction for components of the autophagy pathway. We detected LC3-II protein (an autophagy marker) as well as Rab11 protein, a component of the endosomal pathway. We also observed that the virion-containing vesicles were single-walled; thus, they are not autophagosomes. These results suggested that some VZ virions after secondary envelopment were transported to the outer cell membrane in a vesicle derived from both the autophagy and endosomal pathways, such as an amphisome. Thus, these results demonstrate that herpesvirus trafficking pathways can converge with the autophagy pathway. Full article

(This article belongs to the Special Issue Herpesviruses)

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