Current In Vitro Models to Study Varicella Zoster Virus Latency and Reactivation.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that causes varicella (chicken pox) during primary infection and establishes latency in peripheral neurons. Symptomatic reactivation often presents as zoster (shingles), but it has also been linked to life-threatening diseases such as encephalitis, vasculopathy and meningitis. Zoster may be followed by postherpetic neuralgia, neuropathic pain lasting after resolution of the rash. The mechanisms of varicella zoster virus (VZV) latency and reactivation are not well characterized. This is in part due to the human-specific nature of VZV that precludes the use of most animal and animal-derived neuronal models. Recently, in vitro models of VZV latency and reactivation using human neurons derived from stem cells have been established facilitating an understanding of the mechanisms leading to VZV latency and reactivation. From the models, c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase (PI3K) and nerve growth factor (NGF) have all been implicated as potential modulators of VZV latency/reactivation. Additionally, it was shown that the vaccine-strain of VZV is impaired for reactivation. These models may also aid in the generation of prophylactic and therapeutic strategies to treat VZV-associated pathologies. This review summarizes and analyzes the current human neuronal models used to study VZV latency and reactivation, and provides some strategies for their improvement.

2018 Colorado Alphaherpesvirus Latency Society Symposium.

Meeting Report on the 8th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS), held on May 16-19, 2018, in Vail, Colorado.

Interleukin-6 and type 1 interferons inhibit varicella zoster virus replication in human neurons.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that, following primary infection (varicella), establishes latency in sensory, autonomic, sympathetic and parasympathetic neurons, where it remains until reactivation (zoster). VZV-specific cell-mediated immune responses maintain VZV latency; thus, immunosuppressed and elderly persons are at risk of reactivation and associated neurological diseases. However, the cytokines produced by the immune system that control VZV in neurons are largely unknown. Therefore, to better understand how the immune system may restrict VZV in neurons, we studied interleukin-6, tumor necrosis factor-alpha and type 1 interferons for their ability to inhibit VZV replication in human neurons in vitro. Our studies revealed that VZV transcription and viral spread were significantly reduced by interleukin-6 and type 1 interferons, and to a lesser extent by tumor necrosis factor-alpha. These findings will help in understanding how the innate immune system limits virus replication in neurons in vivo.

Varicella Zoster Virus Induces Nuclear Translocation of the Neurokinin-1 Receptor, Promoting Lamellipodia Formation and Viral Spread in Spinal Astrocytes.

Background: Varicella zoster virus (VZV) can present as a myelopathy with spinal astrocyte infection. Recent studies support a role for the neurokinin-1 receptor (NK-1R) in virus infections, as well as for cytoskeletal alterations that may promote viral spread. Thus, we examined the role of NK-1R in VZV-infected primary human spinal astrocytes (HA-sps) to shed light on the pathogenesis of VZV myelopathy. Methods: Mock- and VZV-infected HA-sps were examined for substance P (subP) production, NK-1R localization, morphological changes, and viral spread in the presence or absence of the NK-1R antagonists aprepitant and rolapitant. Results: VZV infection of HA-sps induced nuclear localization of full-length and truncated NK-1R in the absence of the endogenous ligand, subP, and was associated with extensive lamellipodia formation and viral spread that was inhibited by NK-1R antagonists. Conclusions: We have identified a novel, subP-independent, proviral function of nuclear NK-1R associated with lamellipodia formation and viral spread that is distinct from subP-induced NK-1R cell membrane/cytoplasmic localization without lamellipodia formation. These results suggest that binding of a putative viral ligand to NK-1R produces a dramatically different NK-1R downstream effect than binding of subP. Finally, the NK-1R antagonists aprepitant and rolapitant provide promising alternatives to nucleoside analogs in treating VZV infections, including myelopathy.

Erratum to: 2017 Colorado alphaherpesvirus latency society symposium.

On page 650, the institutional affiliation of Dr. Werner J.D. Ouwendijk was incorrectly listed as Freie Universitaet Berlin. It should instead be Erasmus MC, Rotterdam, The Netherlands.

Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

Induction of varicella zoster virus DNA replication in dissociated human trigeminal ganglia.

Varicella zoster virus (VZV), a human neurotropic alphaherpesvirus, becomes latent after primary infection and reactivates to produce zoster. To study VZV latency and reactivation, human trigeminal ganglia removed within 24 h after death were mechanically dissociated, randomly distributed into six-well tissue culture plates and incubated with reagents to inactivate nerve growth factor (NGF) or phosphoinositide 3-kinase (PI3-kinase) pathways. At 5 days, VZV DNA increased in control and PI3-kinase inhibitor-treated cultures to the same extent, but was significantly more abundant in anti-NGF-treated cultures (p = 0.001). Overall, VZV DNA replication is regulated in part by an NGF pathway that is PI3-kinase-independent.

Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

Alphaherpesvirus DNA replication in dissociated human trigeminal ganglia.

Analysis of the frequency and PCR-quantifiable abundance of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) DNA in multiple biological replicates of cells from dissociated randomly distributed human trigeminal ganglia (TG) of four subjects revealed an increase in both parameters and in both viruses during 5 days of culture, with no further change by 10 days. Dissociated TG provides a platform to analyze initiation of latent virus DNA replication within 5 days of culture.

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene.

UNLABELLED: Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5(P)) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2(P))-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. IMPORTANCE: Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5(P) occupancy decreased and S2(P) levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.

B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.

Interferon Gamma Prolongs Survival of Varicella-Zoster Virus-Infected Human Neurons In Vitro.

Infection of human neurons in vitro with varicella-zoster virus (VZV) at a low multiplicity of infection does not result in a cytopathic effect (CPE) within 14 days postinfection (dpi), despite production of infectious virus. We showed that by 28 dpi a CPE ultimately developed in infected neurons and that interferon gamma inhibited not only the CPE but also VZV DNA accumulation, transcription, and virus production, thereby prolonging the life of VZV-infected neurons.

Inhibition of phosphorylated-STAT1 nuclear translocation and antiviral protein expression in human brain vascular adventitial fibroblasts infected with varicella-zoster virus.

In varicella-zoster virus (VZV)-infected primary human brain vascular adventitial fibroblasts (BRAFs), levels of beta interferon (IFN-ß,) STAT1, and STAT2 transcripts as well as STAT1 and STAT2 protein were decreased. IFN-α transcript levels were increased but not secreted IFN-α protein levels. Compared to IFN-α-treated control results, in VZV-infected BRAFs, phosphorylated STAT1 did not translocate to the nucleus, resulting in impaired downstream expression of interferon-inducible antiviral Mx1. Overall, VZV interference with the type I interferon pathway may promote virus persistence in cerebral arteries.

Varicella zoster virus DNA does not accumulate in infected human neurons.

Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. It is unclear why human neurons infected in vitro with VZV at low multiplicity of infection do not exhibit a cytopathic effect (CPE) even though all VZV genes are transcribed, VZV proteins from all kinetic classes are translated and minimal infectious virus is produced. Here, we show that the lack of VZV-induced CPE correlates with the low abundance of viral DNA.

Comparison of varicella-zoster virus RNA sequences in human neurons and fibroblasts.

Varicella-zoster virus (VZV) infection causes varicella, after which the virus becomes latent in ganglionic neurons. In tissue culture, VZV-infected human neurons remain viable at 2 weeks, whereas fibroblasts develop cytopathology. Next-generation RNA sequencing was used to compare VZV transcriptomes in neurons and fibroblasts and identified only 12 differentially transcribed genes of the 70 annotated VZV open reading frames (ORFs), suggesting that defective virus transcription does not account for the lack of cell death in VZV-infected neurons in vitro.

Varicella zoster virus (VZV)-human neuron interaction.

Varicella zoster virus (VZV) is a highly neurotropic, exclusively human herpesvirus. Primary infection causes varicella (chickenpox), wherein VZV replicates in multiple organs, particularly the skin. Widespread infection in vivo is confirmed by the ability of VZV to kill tissue culture cells in vitro derived from any organ. After varicella, VZV becomes latent in ganglionic neurons along the entire neuraxis. During latency, virus DNA replication stops, transcription is restricted, and no progeny virions are produced, indicating a unique virus-cell (neuron) relationship. VZV reactivation produces zoster (shingles), often complicated by serious neurological and ocular disorders. The molecular trigger(s) for reactivation, and thus the identity of a potential target to prevent it, remains unknown due to an incomplete understanding of the VZV-neuron interaction. While no in vitro system has yet recapitulated the findings in latently infected ganglia, recent studies show that VZV infection of human neurons in SCID mice and of human stem cells, including induced human pluripotent stem cells and normal human neural progenitor tissue-like assemblies, can be established in the absence of a cytopathic effect. Usefulness of these systems in discovering the mechanisms underlying reactivation awaits analyses of VZV-infected, highly pure (>90%), terminally differentiated human neurons capable of prolonged survival in vitro.

Arenavirus infection induces discrete cytosolic structures for RNA replication.

Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus.

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

Interaction domains of the UL16 and UL21 tegument proteins of herpes simplex virus.

The UL16 protein of herpes simplex virus is capsid associated and was previously identified as a binding partner of the membrane-associated UL11 tegument protein (J. S. Loomis, R. J. Courtney, and J. W. Wills, J. Virol. 77:11417-11424, 2003). In those studies, a less-prominent, approximately 65-kDa binding partner of unknown identity was also observed. Mass spectrometry studies have now revealed this species to be UL21, a tegument protein that has been implicated in the transport of capsids in the cytoplasm. The validity of the mass spectrometry results was tested in a variety of coimmunoprecipitation and glutathione S-transferase pull-down experiments. The data revealed that UL21 and UL16 can form a complex in the absence of other viral proteins, even when the assays used proteins purified from Escherichia coli. Moreover, UL11 was able to pull down UL21 only when UL16 was present, suggesting that all three proteins can form a complex. Deletion analyses revealed that the second half of UL21 (residues 268 to 535) is sufficient for the UL16 interaction and packaging into virions; however, attempts to map a subdomain of UL16 were largely unsuccessful, with only the first 40 (of 373) residues being found to be dispensable. Nevertheless, it is clear that UL16 must have two distinct binding sites, because covalent modification of its free cysteines with N-ethylmaleimide blocked binding to UL11 but not UL21. These findings should prove useful for elucidating the molecular machinery used to transmit a signal into a virion when it attaches to cells, a recently discovered mechanism in which UL16 is a central player.