The biological role of viral tRNA-like molecules in a murine gammaherpesvirus infection

Members of the subfamily Gammaherpesvirinae commonly establish latency within lymphoid cells and are associated with lymphoproliferative disease. Gammaherpesviruses include the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpes virus. Due to the narrow host range of infection exhibited by these viruses and their limited productive growth in vitro, the events occurring during lytic replication and the establishment of latency are not well characterised. Murine gammaherpesvirus 68 (MHV-68) is able to undergo productive replication in a number of cell types in vitro and infects laboratory mice; consequently it provides an excellent model for study of gammaherpesvirus infection. MHV-68 encodes eight viral tRNA-like molecules (vtRNAl-8), which resemble cellular tRNAs in that they have a predicted cloverleaf-like secondary structure and are transcribed by RNA polymerase III. However unlike cellular tRNAs they are not amino-acylated and therefore do not function directly during protein synthesis. They are known to be expressed to high levels during both latency and lytic replication. However their role within infection is not known.The aim of this project was to characterise the vtRNAs. The presence of the vtRNAs within purified, RNase treated viral stocks indicated their packaging within the MHV-68 virion. Although both viral and cellular mRNAs were also present, it appeared that the major RNA species packaged by MHV-68 were small RNA molecules, such as the vtRNAs. Incorporation of RNA molecules into the virion is not unique to MHV-68 as other herpesviruses have been found to package RNA, although the vtRNAs represent the only packaged small viral non-coding RNA molecules discovered so far. In addition, this is the first study to demonstrate the preferential incorporation of small RNA molecules by a herpesvirus. The mechanism by which the vtRNAs assemble into the virion is not clear. In situ hybridization demonstrated that within infected cells the vtRNAs localized to globular areas within the nucleus and were also found at high levels within the cytoplasm. Electrophoretic mobility shift assays performed using vtRNAl and vtRNA4 indicated binding to protein complexes present within both the nucleus and cytoplasm of infected cells. Inhibition of vtRNA-protein binding by an anti-MHV-68 antibody indicated direct interaction of the vtRNAs with viral protein(s). Hence it is likely that their incorporation is mediated through binding to viral protein(s) during virion assembly in either the nucleus or cytoplasm.MHV-76 is a deletion mutant of MHV-68, which lacks all eight vtRNAs along with four other genes (M1-M4). The contribution of the vtRNAs to viral pathogenesis has been investigated by construction of recombinant MHV-76, which expressed vtRNAsl-5 under their natural promoters. The insertion of the vtRNAs into MHV-76 had no effect on the ability of the virus to replicate in vitro. In addition, the recombinant viruses displayed identical characteristics to MHV-76 following intranasal infection of BALB/c mice, demonstrated by the levels of lytic virus present within the lung and the levels of latent virus within the spleen. Therefore the role of the vtRNAs within infection remains to be determined and the recombinant viruses produced in this project will provide excellent tools to investigate their function further through both in vitro and in vivo analysis

Kinetics of Facultative Heterochromatin and Polycomb Group Protein Association with the Herpes Simplex Viral Genome during Establishment of Latent Infection

ABSTRACT The herpes simplex virus (HSV) genome is associated with heterochromatic histone modifications, including trimethylation of the lysine 27 residue of histone H3 (H3K27me3), during latent infection of neurons. Here we have examined the kinetics of general chromatin and H3K27me3 association with the viral genome during establishment of latent infection. Using both wild-type virus and a mutant virus that is unable to undergo replication in neurons, we found that histone H3 associates with viral gene promoters by 7 days postinfection (dpi). Levels of H3K27me3 were low at 7 dpi but increased dramatically by 14 dpi. Hence, general chromatin association and/or other factors may play a key role(s) in the initial silencing of lytic genes, and H3K27me3 may play a role in further suppression of the genome and/or the maintenance of latency. A component of Polycomb repressive complex 2 (PRC2), which mediates the addition of K27me3 to histone H3 (Suz12), was also recruited by 14 dpi. We have shown previously that the levels of H3K27me3 during latent infection are increased in the presence of the latency-associated transcript (LAT). However, the initial targeting of PRC2 was not found to be dependent on the LAT. We found that a component of the PRC1 complex (Bmi1), which binds to H3K27me3, was not enriched at promoters found previously to be enriched for H3K27me3. Our results are consistent with (i) chromatinization of viral DNA or other mechanisms causing the initial silencing of HSV lytic genes and (ii) facultative heterochromatin maintaining that silencing during latent infection of neurons

Role for A-Type Lamins in Herpesviral DNA Targeting and Heterochromatin Modulation

Posttranslational modification of histones is known to regulate chromatin structure and transcriptional activity, and the nuclear lamina is thought to serve as a site for heterochromatin maintenance and transcriptional silencing. In this report, we show that the nuclear lamina can also play a role in the downregulation of heterochromatin and in gene activation. Herpes simplex virus DNA initiates replication in replication compartments near the inner edge of the nucleus, and histones are excluded from these structures. To define the role of nuclear lamins in HSV replication, we examined HSV infection in wild-type and A-type lamin–deficient (Lmna−/−) murine embryonic fibroblasts (MEFs). In Lmna−/− cells, viral replication compartments are reduced in size and fail to target to the nuclear periphery, as observed in WT cells. Chromatin immunoprecipitation and immunofluorescence studies demonstrate that HSV DNA is associated with increased heterochromatin in Lmna−/− MEFs. These results argue for a functional role for A-type lamins as viral gene expression, DNA replication, and growth are reduced in Lmna−/− MEFs, with the greatest effect on viral replication at low multiplicity of infection. Thus, lamin A/C is required for targeting of the viral genome and the reduction of heterochromatin on viral promoters during lytic infection. The nuclear lamina can serve as a molecular scaffold for DNA genomes and the protein complexes that regulate both euchromatin and heterochromatin histone modifications

Variation in the helminth community structure in spiny mice (Acomys dimidiatus) from four montane wadis in the St Katherine region of the Sinai Peninsula in Egypt

We compared helminth communities in spiny mice (Acomys dimidiatus) from 4 wadis in the arid montane region of the southern Sinai in Egypt, in a 4-week period in late summer. Total helminth species richness was 14 (8 nematodes, 5 cestodes and 1 acanthocephalan) with 94% of mice carrying at least 1 species and an overall mean species richness of 1.85. The most prevalent parasites were Protospirura muricola (47.8%) and Dentostomella kuntzi (46.3%). One larval cestode, Joyeuxiella rossicum, represents a new host record. The helminth community was dominated by intestinal nematodes (88.7%) of which 58.2% were arthropod-transmitted heteroxenic species. At the component community level, 70% of the worms were recovered from mice in just two wadis (Gharaba and Tlah) and 48.6% of intestinal nematodes were from Wadi Gharaba. Although only 7 species of helminths were recorded from Wadi Gharaba, this site gave the highest Berger-Parker dominance index because P. muricola. P. muricola was also dominant in Wadi El Arbaein whilst Syphacia minuta was the dominant species in Wadis Gebal and Tlah. At the infracommunity level, mean species richness and Brillouin’s index of diversity were highest in Wadi Tlah and lowest in Wadi Gebal, and the former was age dependent. Whilst mice from different wadis differed in the nematodes that were most common, those from Wadi Gharaba carried the highest mean number of worms/mouse. The abundance of P. muricola in particular varied markedly between sites: Wadi Gharaba was distinct as the site showing the highest mean worm burden whereas mice from Wadi Gebal were uninfected. None of the directly transmitted oxyuroid nematodes showed significant variation in abundance between wadis, or host sex or age classes. Overall, the single extrinsic factor in the study, site of capture, was more important than the intrinsic factors in explaining variation in helminth communities in the region. We conclude that in the high mountains of southern Sinai, each wadi is distinct in terms of its rodent parasites, and hence we expect spatially different coevolutionary pressures on their hosts, with resultant variation in life-histories

Risk and protective factors for self-harm and suicide in children and adolescents: a systematic review and meta-analysis protocol.

INTRODUCTION Self-harm and suicide are major public health concerns among children and adolescents. Many risk and protective factors for suicide and self-harm have been identified and reported in the literature. However, the capacity of these identified risk and protective factors to guide assessment and management is limited due to their great number. This protocol describes an ongoing systematic review and meta-analysis which aims to examine longitudinal studies of risk factors for self-harm and suicide in children and adolescents, to provide a comparison of the strengths of association of the various risk factors for self-harm and suicide and to shed light on those that require further investigation. METHODS AND ANALYSIS We perform a systematic search of the literature using the databases EMBASE, PsycINFO, Medline, CINAHL and HMIC from inception up to 28 October 2020, and the search will be updated before the systematic review publication. Additionally, we will contact experts in the field, including principal investigators whose peer-reviewed publications are included in our systematic review as well as investigators from our extensive research network, and we will search the reference lists of relevant reviews to retrieve any articles that were not identified in our search. We will extract relevant data and present a narrative synthesis and combine the results in meta-analyses where there are sufficient data. We will assess the risk of bias for each study using the Newcastle-Ottawa Scale and present a summary of the quantity and the quality of the evidence for each risk or protective factor. ETHICS AND DISSEMINATION Ethical approval will not be sought as this is a systematic review of the literature. Results will be published in mental health journals and presented at conferences focused on suicide prevention. PROSPERO REGISTRATION NUMBER CRD42021228212

Single-genome analysis reveals heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity

Neuronal Stress Pathway Mediating a Histone Methyl/Phospho Switch Is Required for Herpes Simplex Virus Reactivation

Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK towards stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency

Neuronal hyperexcitability is a DLK-dependent trigger of Herpes Simplex Virus reactivation that can be induced by IL-1

Herpes simplex virus-1 (HSV-1) establishes a latent infection in neurons and periodically reactivates to cause disease. The stimuli that trigger HSV-1 reactivation have not been fully elucidated. We demonstrate HSV-1 reactivation from latently infected mouse neurons induced by forskolin requires neuronal excitation. Stimuli that directly induce neurons to become hyperexcitable also induced HSV-1 reactivation. Forskolin-induced reactivation was dependent on the neuronal pathway of DLK/JNK activation and included an initial wave of viral gene expression that was independent of histone demethylase activity and linked to histone phosphorylation. IL-1β is released under conditions of stress, fever and UV exposure of the epidermis; all known triggers of clinical HSV reactivation. We found that IL-1β induced histone phosphorylation and increased the excitation in sympathetic neurons. Importantly, IL-1β triggered HSV-1 reactivation, which was dependent on DLK and neuronal excitability. Thus, HSV-1 co-opts an innate immune pathway resulting from IL-1 stimulation of neurons to induce reactivation

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort

Virology—the path forward

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US