Herpes Simplex Virus 1 Replication, Ocular Disease, and Reactivations from Latency Are Restricted Unilaterally after Inoculation of Virus into the Lip.

Ocular herpes simplex keratitis (HSK) is a consequence of viral reactivations from trigeminal ganglia (TG) and occurs almost exclusively in the same eye in humans. In our murine oro-ocular (OO) model, herpes simplex virus 1 (HSV-1) inoculation in one side of the lip propagates virus to infect the ipsilateral TG. Replication here allows infection of the brainstem and infection of the contralateral TG. Interestingly, HSK was observed in our OO model only from the eye ipsilateral to the site of lip infection. Thus, unilateral restriction of HSV-1 may be due to differential kinetics of virus arrival in the ipsilateral versus contralateral TG. We inoculated mice with HSV-1 reporter viruses and then superinfected them to monitor changes in acute- and latent-phase gene expression in TG after superinfection compared to the control (single inoculation). Delaying superinfection by 4 days after initial right lip inoculation elicited failed superinfecting-virus gene expression and eliminated clinical signs of disease. Initial inoculation with thymidine kinase-deficient HSV-1 (TKdel) completely abolished reactivation of wild-type (WT) superinfecting virus from TG during the latent stage. In light of these seemingly failed infections, viral genome was detected in both TG. Our data demonstrate that inoculation of HSV-1 in the lip propagates virus to both TG, but with delay in reaching the TG contralateral to the side of lip infection. This delay is responsible for restricting viral replication to the ipsilateral TG, which abrogates ocular disease and viral reactivations from the contralateral side. These observations may help to understand why HSK is observed unilaterally in humans, and they provide insight into vaccine strategies to protect against HSK.IMPORTANCE Herpetic keratitis (HK) is the leading cause of blindness by an infectious agent in the developed world. This disease can occur after reactivation of herpes simplex virus 1 in the trigeminal ganglia, leading to dissemination of virus to, and infection of, the cornea. A clinical paradox is evidenced by the bilateral presence of latent viral genomes in both trigeminal ganglia, while for any given patient the disease is unilateral with recurrences in a single eye. Our study links the kinetics of early infection to unilateral disease phenomenon and demonstrates protection against viral reactivation when kinetics are exploited. Our results have direct implications in the understanding of human disease pathogenesis and immunotherapeutic strategies for the treatment of HK and viral reactivations.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment.

Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely "multiple-acute"). Viral genomes in the "multiple-acute" pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the "multiple-latency" pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.

Functional evidence for the involvement of microtubules and dynein motor complexes in TRIM5α-mediated restriction of retroviruses.

UNLABELLED: The tripartite motif (TRIM) family of proteins includes the TRIM5α antiretroviral restriction factor. TRIM5α from many Old World and some New World monkeys can restrict the human immunodeficiency virus type 1 (HIV-1), while human TRIM5α restricts N-tropic murine leukemia virus (N-MLV). TRIM5α forms highly dynamic cytoplasmic bodies (CBs) that associate with and translocate on microtubules. However, the functional involvement of microtubules or other cytoskeleton-associated factors in the viral restriction process had not been shown. Here, we demonstrate the dependency of TRIM5α-mediated restriction on microtubule-mediated transport. Pharmacological disruption of the microtubule network using nocodazole or disabling it using paclitaxel (originally named taxol) decreased the restriction of N-MLV and HIV-1 by human or simian alleles of TRIM5α, respectively. In addition, pharmacological inhibition of dynein motor complexes using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and small interfering RNA-mediated depletion of the dynein heavy chain (DHC) similarly decreased TRIM5α-mediated restriction. The loss in restriction resulting from either the disassembly of microtubules or the disruption of dynein motor activity was seen for both endogenous and overexpressed TRIM5α and was not due to differences in protein stability or cell viability. Both nocodazole treatment and DHC depletion interfered with the dynamics of TRIM5α CBs, increasing their size and altering their intracellular localization. In addition, nocodazole, paclitaxel, and DHC depletion were all found to increase the stability of HIV-1 cores in infected cells, providing an alternative explanation for the decreased restriction. In conclusion, association with microtubules and the translocation activity of dynein motor complexes are required to achieve efficient restriction by TRIM5α. IMPORTANCE: The primate innate cellular defenses against infection by retroviruses include a protein named TRIM5α, belonging to the family of restriction factors. TRIM5α is present in the cytoplasm, where it can intercept incoming retroviruses shortly after their entry. How TRIM5α manages to be present at the appropriate subcytoplasmic location to interact with its target is unknown. We hypothesized that TRIM5α, either as a soluble protein or a high-molecular-weight complex (the cytoplasmic body), is transported within the cytoplasm by a molecular motor called the dynein complex, which is known to interact with and move along microtubules. Our results show that destructuring microtubules or crippling their function decreased the capacity of human or simian TRIM5α to restrict their retroviral targets. Inhibiting dynein motor activity, or reducing the expression of a key component of this complex, similarly affected TRIM5α-mediated restriction. Thus, we have identified specific cytoskeleton structures involved in innate antiretroviral defenses.

Use and persistence of single and multiple inhaler triple therapy prescribed for patients with COPD in France: a retrospective study on THIN database (OPTI study).

INTRODUCTION: From 2018 single inhaler triple therapy (SITT) became available in France to treat moderate-to-severe chronic obstructive pulmonary disease (COPD). Given its simplified inhaler use compared with multiple inhaler triple therapy (MITT), this therapeutic option has the potential to offer benefit in terms of improved persistence and adherence. Given the lack of real-world evidence of the effectiveness of triple therapy, this study was designed to evaluate the use of MITT and SITT in France and compare persistence. METHODS: A retrospective cohort study was performed. Patients with COPD who initiated triple therapy between 1 July 2017 and 31 December 2019 were included from The Health Improvement Network, a large electronic medical database in France, which includes pharmacy data. A 60-day treatment gap defined discontinuation and thereby persistence. RESULTS: A total of 3134 patients initiated triple therapy for COPD in the study period, among them 485 with SITT. In 2019, the rate of use of SITT was 28.2%. The mean age (67.3 years) and sex (44.2% female) of patients initiating triple therapy was similar between MITT and SITT, and most patients had escalated from dual therapy (84.1%). However, SITT was more frequently initiated by a pulmonologist (59.8%) and a higher prevalence of comorbid asthma was observed for SITT (47.0% vs 37.9%). Persistence was assessed among patients who did not discontinue after a single dispensation of triple therapy (n=1674). Median persistence was 181 days for SITT and 135 days for MITT, and the covariate-adjusted HR for persistence was 1.47 (p<0.001) and the estimated persistence at 1 year was 33% for SITT compared with 18% for MITT. DISCUSSION: This study suggests that persistence was higher for the patients treated with SITT compared with MITT in France. Moreover, most patients initiated with triple therapy were previously treated with dual therapy and had exacerbations in the previous year.