Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3+CD4+, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.
Differential Impacts of HHV-6A versus HHV-6B Infection in Differentiated Human Neural Stem Cells
Within the family Herpesviridae , sub-family β-herpesvirinae , and genus Roseolovirus , there are only three human herpesviruses that have been described: HHV-6A, HHV-6B, and HHV-7. Initially, HHV-6A and HHV-6B were considered as two variants of the same virus (i.e., HHV6). Despite high overall genetic sequence identity (~90%), HHV-6A and HHV-6B are now recognized as two distinct viruses. Sequence divergence (e.g., >30%) in key coding regions and significant differences in physiological and biochemical profiles (e.g., use of different receptors for viral entry) underscore the conclusion that HHV-6A and HHV-6B are distinct viruses of the β-herpesvirinae . Despite these viruses being implicated as causative agents in several nervous system disorders (e.g., multiple sclerosis, epilepsy, and chronic fatigue syndrome), the mechanisms of action and relative contributions of each virus to neurological dysfunction are unclear. Unresolved questions regarding differences in cell tropism, receptor use and binding affinity (i.e., CD46 versus CD134), host neuro-immunological responses, and relative virulence between HHV-6A versus HHV-6B prevent a complete characterization. Although it has been shown that both HHV-6A and HHV-6B can infect glia (and, recently, cerebellar Purkinje cells), cell tropism of HHV-6A versus HHV-6B for different nerve cell types remains vague. In this study, we show that both viruses can infect different nerve cell types (i.e., glia versus neurons) and different neurotransmitter phenotypes derived from differentiated human neural stem cells. As demonstrated by immunofluorescence, HHV-6A and HHV-6B productively infect VGluT1-containing cells (i.e., glutamatergic neurons) and dopamine-containing cells (i.e., dopaminergic neurons). However, neither virus appears to infect GAD67-containing cells (i.e., GABAergic neurons). As determined by qPCR, expression of immunological factors (e.g., cytokines) in cells infected with HHV-6A versus HHV6-B also differs. These data along with morphometric and image analyses of infected differentiated neural stem cell cultures indicate that while HHV-6B may have greater opportunity for transmission, HHV-6A induces more severe cytopathic effects (e.g., syncytia) at the same post-infection end points. Cumulatively, results suggest that HHV-6A is more virulent than HHV-6B in susceptible cells, while neither virus productively infects GABAergic cells. Consistency between these in vitro data and in vivo experiments would provide new insights into potential mechanisms for HHV6-induced epileptogenesis.
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- NSF-PAR ID:
- 10396376
- Date Published:
- Journal Name:
- Frontiers in Immunology
- Volume:
- 13
- ISSN:
- 1664-3224
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract While hundreds of thousands of human whole genome sequences (WGS) have been collected in the effort to better understand genetic determinants of disease, these whole genome sequences have less frequently been used to study another major determinant of human health: the human virome. Using the unmapped reads from WGS of over 1000 families, we present insights into the human blood DNA virome, focusing particularly on human herpesvirus (HHV) 6A, 6B, and 7. In addition to extensively cataloguing the viruses detected in WGS of human whole blood and lymphoblastoid cell lines, we use the family structure of our dataset to show that household drives transmission of several viruses, and identify the Mendelian inheritance patterns characteristic of inherited chromsomally integrated human herpesvirus 6 (iciHHV-6). Consistent with prior studies, we find that 0.6% of our dataset’s population has iciHHV, and we locate candidate integration sequences for these cases. We document genetic diversity within exogenous and integrated HHV species and within integration sites of HHV-6. Finally, in the first observation of its kind, we present evidence that suggests widespread de novo HHV-6B integration and HHV-7 integration and reactivation in lymphoblastoid cell lines. These findings show that the unmapped read space of WGS is a promising source of data for virology research.
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ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.more » « less
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INTRODUCTION Balance between excitatory and inhibitory neuron (interneuron) populations in the cortex promotes normal brain function. Interneurons are primarily generated in the medial, caudal, and lateral ganglionic eminences (MGE, CGE, and LGE) of the ventral embryonic forebrain; these subregions give rise to distinct interneuron subpopulations. In rodents, the MGE generates cortical interneurons, the parvalbumin + (PV + ) and somatostatin + (SST + ) subtypes that connect with excitatory neurons to regulate their activity. Defects in interneuron production have been implicated in neurodevelopmental and psychiatric disorders including autism, epilepsy, and schizophrenia. RATIONALE How does the human MGE (hMGE) produce the number of interneurons required to populate the forebrain? The hMGE contains progenitor clusters distinct from what has been observed in the rodent MGE and other germinal zones of the human brain. This cytoarchitecture could be the key to understanding interneuron neurogenesis. We investigated the cellular and molecular properties of different compartments within the developing hMGE, from 14 gestational weeks (GW) to 39 GW (term), to study their contribution to the production of inhibitory interneurons. We developed a xenotransplantation assay to follow the migration and maturation of the human interneurons derived from this germinal region. RESULTS Within the hMGE, densely packed aggregates (nests) of doublecortin + (DCX + ) and LHX6 + cells were surrounded by nestin + progenitor cells and their processes. These DCX + cell–enriched nests (DENs) were observed in the hMGE but not in the adjacent LGE. We found that cells within DENs expressed molecular markers associated with young neurons, such as DCX, and polysialylated neural cell adhesion molecule (PSA-NCAM). A subpopulation also expressed Ki-67, a marker of proliferation; therefore, we refer to these cells as neuroblasts. A fraction of DCX + cells inside DENs expressed SOX2 and E2F1, transcription factors associated with progenitor and proliferative properties. More than 20% of DCX + cells in the hMGE were dividing, specifically within DENs. Proliferating neuroblasts in DENs persisted in the hMGE throughout prenatal human brain development. The division of DCX + cells was confirmed by transmission electron microscopy and time-lapse microscopy. Electron microscopy revealed adhesion contacts between cells within DENs, providing multiple sites to anchor DEN cells together. Neuroblasts within DENs express PCDH19, and nestin + progenitors surrounding DENs express PCDH10; these findings suggest a role for differential cell adhesion in DEN formation and maintenance. When transplanted into the neonatal mouse brain, dissociated hMGE cells reformed DENs containing proliferative DCX + cells, similar to DENs observed in the prenatal human brain. This suggests that DENs are generated by cell-autonomous mechanisms. In addition to forming DENs, transplanted hMGE-derived neuroblasts generated young neurons that migrated extensively into cortical and subcortical regions in the host mouse brain. One year after transplantation, these neuroblasts had differentiated into distinct γ-aminobutyric acid–expressing (GABAergic) interneuron subtypes, including SST + and PV + cells, that showed morphological and functional maturation. CONCLUSION The hMGE harbors DENs, where cells expressing early neuronal markers continue to divide and produce GABAergic interneurons. This MGE-specific arrangement of neuroblasts in the human brain is present until birth, supporting expanded neurogenesis for inhibitory neurons. Given the robust neurogenic output from this region, knowledge of the mechanisms underlying cortical interneuron production in the hMGE will provide insights into the cell types and developmental periods that are most vulnerable to genetic or environmental insults. Nests of DCX + cells in the ventral prenatal brain. Schematic of a coronal view of the embryonic human forebrain showing the medial ganglionic eminence (MGE, green), with nests of DCX + cells (DENs, green). Nestin + progenitor cells (blue) are present within the VZ and iSVZ and are intercalated in the oSVZ (where DENs reside). The initial segment of the oSVZ contains palisades of nestin + progenitors referred to as type I clusters (light blue cells) around DENs. In the outer part of the oSVZ, DENs transition to chains of migrating DCX + cells; surrounding nestin + progenitors are arranged into groups of cells referred to as type II clusters (white cells). In addition to proliferation of nestin + progenitors, cell division is present among DCX + cells within DENs, suggesting multiple progenitor states for the generation of MGE-derived interneurons in the human forebrain. ILLUSTRATION: NOEL SIRIVANSANTImore » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fimmu.2022.847106
