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Manabu Akazawa,1 Takahiro Sato,2 Nozomi Ebata,2 Michael LoPresti,3 Ryosuke Nishi3 1Department of Public Health and Epidemiology, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan; 2Medical Affairs, Rare Disease, Pfizer Japan Inc, Shibuya-ku, Tokyo, Japan; 3Value & Access, INTAGE Healthcare Inc., Chiyoda-ku, Tokyo, JapanCorrespondence: Takahiro Sato, Pfizer Japan Inc, 3-22-7 Yoyogi, Shibuya-ku, Tokyo, 151-8589, Japan, Tel +81 90-2935-0848, Email takahiro.sato@pfizer.comBackground: About one-third of caregivers of pediatric or adolescent growth hormone deficiency (pGHD) patients in Japan have reported poor treatment adherence. However, few studies have examined factors related to adherence for that group.Objective: The aim of this study is to consider factors related to poor adherence to daily treatment among caregivers of pGHD patients in Japan.Methods: A cross-sectional survey was conducted among caregivers of pGHD patients in Japan. Caregivers were asked about demographic and treatment characteristics, health literacy, treatment satisfaction, opinions about treatment, and treatment adherence. Health literacy was assessed using the 14-item health literacy scale (HLS-14). Adherence was assessed using the 8-item Morisky Medication Adherence Scale (MMAS-8). Statistical association with adherence was considered using Chi-square and Student’s t–testing. An exploratory factor analysis (EFA) and K-means cluster analysis was conducted to consider the influence of treatment satisfaction and opinions concerning treatment on adherence.Results: Responses were collected from 112 caregivers. The caregiver’s age being 30– 39 years old, the primary caregiver being male, the primary caregiver being employed, and low functional health literacy for the caregiver were associated with poor adherence. Patients being pre-elementary school age was also associated with poor adherence. Low satisfaction with drug treatment and/or their device and communication with healthcare professionals (HCPs), and lack of agreement with the importance of treatment management (eg, keeping injection records, getting informed about the disease/therapy, reporting non-adherence, and sticking to an administration schedule), were also associated with poor adherence.Conclusion: Strategies to improve treatment adherence among caregivers of pGHD patients in Japan should consider the age, gender, and employment status of the caregiver – as well as their functional literacy. Improvement in satisfaction with the drug or device used, better communication with HCPs, and greater awareness of the importance of treatment management, may also lead to better adherence.Keywords: growth hormone, adherence, determinants, satisfaction, communication, health literacy

Manabu Akazawa,1 Daisuke Shima,2 Takahiro Sato,2 Emi Shoji,2 Michael LoPresti,3 Ryosuke Nishi3 1Department of Public Health and Epidemiology, Meiji Pharmaceutical University, Tokyo, Japan; 2Medical Affairs, Rare Disease, Pfizer Japan Inc, Tokyo, Japan; 3Market Access, INTAGE Healthcare Inc, Tokyo, JapanCorrespondence: Takahiro Sato, Medical Affairs, Rare Disease, Pfizer Japan Inc, 3-22-7 Yoyogi, Shibuya-ku, Tokyo, 151-8589, Japan, Tel +81 90-2935-0848, Email takahiro.sato@pfizer.comBackground: Poor adherence to daily human growth hormone (hGH) treatment has been shown to be associated with poor clinical outcomes for growth hormone deficiency (GHD) patients. However, few studies have examined the perception of adherence to hGH treatment among both physicians and caregivers in Japan.Objective: The aim of this study is to examine the perception of adherence for daily hGH treatment among physicians and caregivers of pediatric and adolescent patients treated with GH in Japan. Moreover, we explore reasons for skipping treatment and the potential impact of a once-weekly treatment on adherence.Methods: A cross-sectional survey was conducted in Japan among physicians that prescribe daily hGH treatment and caregivers that have administered daily hGH treatment to children/adolescents for 3 months or longer. The Morisky Medication Adherence Scale (MMAS-8) was used to gauge perceived adherence for both physician and caregiver groups. Caregivers were also questioned regarding reasons for missing injections. Moreover, both groups were asked about the impact of a once-weekly treatment on adherence.Results: Responses were collected from 123 physicians and 112 caregivers. Physicians reported that 18.1% of patients have poor adherence based on the MMAS-8 instrument. In contrast, 32.1% of the caregivers reported poor adherence. “Simply forgetting”, “Patient refused/resisted”, and being “Busy with school activities, etc” were the most commonly selected reasons by caregivers for missing an injection. Physicians felt that a once-weekly injection could improve adherence for 64.5% of patients with poor adherence. Moreover, 56.9% of the caregivers that reported an experience of missed injections felt that a once-weekly injection would improve their adherence.Conclusion: Approaches to improve adherence to hGH treatment in Japan are continuously needed. While further research is needed to understand factors most likely to improve adherence, availability of a once-weekly treatment is expected to help improve adherence.Keywords: adherence, growth hormone, Japanese, recombinant, injections, MMAS

Abstract Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The ‘dark proteome’ includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.

RNA granules are cytoplasmic, non-membranous ribonucleoprotein compartments that form ubiquitously and are often referred to as foci for post-transcriptional gene regulation. Recent research on RNA processing bodies (PB) and stress granules (SG) has shown wide implications of these cytoplasmic RNA granules and their components in suppression of RNA translation as host intracellular innate immunity against infecting viruses. Many RNA viruses either counteract or co-opt these RNA granules; however, many fundamental questions about DNA viruses with respect to their interaction with these two RNA granules remain elusive. Kaposi’s sarcoma-associated herpesvirus (KSHV), a tumor-causing DNA virus, exhibits two distinct phases of infection and encodes ∼90 viral gene products during the lytic phase of infection compared to only a few (∼5) during the latent phase. Thus, productive KSHV infection relies heavily on the host cell translational machinery, which often links to the formation of PB and SG. One major question is how KSHV counteracts the hostile environment of RNA granules for its productive infection. Recent studies demonstrated that KSHV copes with the translational suppression by cellular RNA granules, PB and SG, by expressing ORF57, a viral RNA-binding protein, during KSHV lytic infection. ORF57 interacts with Ago2 and GW182, two major components of PB, and prevents the scaffolding activity of GW182 at the initial stage of PB formation in the infected cells. ORF57 also interacts with protein kinase R (PKR) and PKR-activating protein (PACT) to block PKR dimerization and kinase activation, and thus inhibits eIF2α phosphorylation and SG formation. The homologous immediate-early regulatory protein ICP27 of herpes simplex virus type 1 (HSV-1), but not the EB2 protein of Epstein-Barr virus (EBV), shares this conserved inhibitory function with KSHV ORF57 on PB and SG. Through KSHV ORF57 studies, we have learned much about how a DNA virus in the infected cells is equipped to evade host antiviral immunity for its replication and productive infection. KSHV ORF57 would be an excellent viral target for development of anti-KSHV-specific therapy.

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi's sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.

Protein lysine methyltransferases (PKMTs) methylate histone and non-histone proteins to regulate biological outcomes such as development and disease including viral infection. While PKMTs have been extensively studied for modulating the antiviral responses via host gene regulation, their role in methylation of proteins encoded by viruses and its impact on host-pathogen interactions remain poorly understood. In this study, we discovered distinct nucleo-cytoplasmic form of euchromatic histone methyltransferase 1 (EHMT1N/C), a PKMT, that phase separates into viral inclusion bodies (IBs) upon cytoplasmic RNA-virus infection (Sendai Virus). EHMT1N/C interacts with cytoplasmic EHMT2 and methylates SeV-Nucleoprotein upon infection. Elevated nucleoprotein methylation during infection correlated with coalescence of small IBs into large mature platforms for efficient replication. Inhibition of EHMT activity by pharmacological inhibitors or genetic depletion of EHMT1N/C reduced the size of IBs with a concomitant reduction in replication. Additionally, we also found that EHMT1 condensation is not restricted to SeV alone but was also seen upon pathogenic RNA viral infections caused by Chandipura and Dengue virus. Collectively, our work elucidates a new mechanism by which cytoplasmic EHMT1 acts as proviral host factor to regulate host-pathogen interaction.

Abstract This article provides a summary of a previously published paper: Terminal Complement Inhibitor Ravulizumab in Generalized Myasthenia Gravis. The paper reported the results of the CHAMPION-MG trial which investigated the drug ravulizumab in the rare disease, myasthenia gravis. Terminal Complement Inhibitor Ravulizumab in Generalized Myasthenia Gravis (MP4 594600 KB)