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The article presents a study which investigated various mouse models to understand human T lymphotropic virus type-I-associated (HTLV-I-associated) adult T-cell leukemia. It states that the ability to manipulate transgenic mice led to more understanding of viral genes responsible for lymphocyte transformation. The pathologic and clinical characteristics of HTLV-I-associated diseases in human are explained.

CD4+ T-cell tines with specificity for individual measles virus (MV) structural proteins were obtained from immunized Lewis rats. Isolated viral proteins, either purified from virions or bacterially expressed were used as antigens for immunological assays. All the cell lines secreted interferon- gamma (IFN-γ) and interleukin-2 (IL-2), but were only weakly cytotoxic to autologous MV-infected astrocytes. When cultured together with memory splenic B lymphocytes these T cells did not induce secretion of MV-specific antibodies. The in viva function of the T-cell lines was investigated in our MV-encephalitis model in the Lewis rat. Within 24 hr of intracerebral infection, adoptive transfer of single MV protein-specific T cells either decreased or prevented the subsequent clinical and histological disease depending on the MV-protein specificity of the cell lines. Furthermore, there was an earlier and enhanced viral clearance from the CNS, without a change in the anti-MV antibody titres of serum and cerebrospinal fluid (CSF) of the recipients and the control-infected animals. Prior depletion of CD8+ T lymphocytes in the recipient animals did not abrogate the protection conferred by CD4+ T-cell lines, indicating that the acute viral CNS disease is being efficiently controlled by virus-specific CD4+ T cells. [ABSTRACT FROM AUTHOR]

Abstract Chronic inflammation has long been associated with a wide range of malignancies, is now widely accepted as a risk factor for development of cancer, and has been implicated as a promoter of a variety of cancers including hematopoietic malignancies. We have described a mouse model uniquely suited to examine the link between inflammation and lymphoma in which the Tax oncogene, expressed in activated T and NK cells, perpetuates chronic inflammation that begins as microscopic intraepithelial lesions and develops into inflammatory nodules, subcutaneous tumors, and large granular lymphocytic leukemia. The use of bioluminescent imaging in these mice has expanded our ability to interrogate aspects of inflammation and tumorigenesis non-invasively. Here we demonstrate that bioluminescence induction in these mice correlated with inflammation resulting from wounding, T cell activation, and exposure to chemical agents. In experiments in which long-term effects of inflammation on disease outcome were monitored, the development of lymphoma was promoted by an inflammatory stimulus. Finally we demonstrated that activation of T-cells in T-cell receptor (TCR) transgenic TAX-LUC animals dramatically exacerbated the development of subcutaneous TCR- CD16+ LGL tumors. The role of activated T-cells and acquired immunity in inflammation-associated cancers is broadly applicable to hematopoietic malignancies, and we propose these mice will be of use in dissecting mechanisms by which activated T-cells promote lymphomagenesis in vivo.

Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo , reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread., Importance: Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection., Competing Interests: K.S., M.P., and A.M. are listed as inventors on a provisional patent related to the work in this article.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission., Competing Interests: The authors declare no conflict of interest.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo., Competing Interests: The authors declare no competing interests that could be perceived to bias this work., (Copyright: © 2023 Maksimova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of "humanized" mice has become a prominent tool for translational animal studies of human diseases. Immunodeficient mice can be humanized by injections of human umbilical cord stem cells. The engraftment of these cells and their development into human lymphocytes has been made possible by the development of novel severely immunodeficient mouse strains. Proven protocols for the generation and analysis of humanized mice in the NSG mouse background are presented here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human umbilical stem cell engraftment of neonatal immunodeficient mice Basic Protocol 2: Human umbilical stem cell engraftment of 4-week-old immunodeficient mice Support Protocol 1: Preparation of human umbilical stem cells Support Protocol 2: Submandibular blood collection from humanized mice and analysis of peripheral blood via flow cytometry., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Human T-cell leukemia virus type 1 (HTLV-1) is the infectious cause of adult T-cell leukemia/lymphoma (ATL), an extremely aggressive and fatal malignancy of CD4 + T-cells. Due to the chemotherapy-resistance of ATL and the absence of long-term therapy regimens currently available for ATL patients, there is an urgent need to characterize novel therapeutic targets against this disease. Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT enzyme that is directly involved in the pathogenesis of multiple different lymphomas through the transcriptional regulation of relevant oncogenes. Recently, our group identified that PRMT5 is overexpressed in HTLV-1-transformed T-cell lines, during the HTLV-1-mediated T-cell immortalization process, and in ATL patient samples. The objective of this study was to determine the importance of PRMT5 on HTLV-1 infected cell viability, T-cell transformation, and ultimately disease induction. Inhibition of PRMT5 enzymatic activity with a commercially available small molecule inhibitor (EPZ015666) resulted in selective in vitro toxicity of actively proliferating and transformed T-cells. EPZ015666-treatment resulted in a dose-dependent increase in apoptosis in HTLV-1-transformed and ATL-derived cell lines compared to uninfected Jurkat T-cells. Using a co-culture model of infection and immortalization, we found that EPZ015666 is capable of blocking HTLV-1-mediated T-cell immortalization in vitro , indicating that PRMT5 enzymatic activity is essential for the HTLV-1 T-cell transformation process. Administration of EPZ015666 in both NSG xenograft and HTLV-1-infected humanized immune system (HIS) mice significantly improved survival outcomes. The cumulative findings of this study demonstrate that the epigenetic regulator PRMT5 is critical for the survival, transformation, and pathogenesis of HTLV-1, illustrating the value of this cellular enzyme as a potential therapeutic target for the treatment of ATL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ernzen, Melvin, Yu, Phelps, Niewiesk, Green and Panfil.)

Respiratory syncytial virus is an important cause of pneumonia in children, the elderly, and immunocompromised individuals. The attachment (G) protein of RSV generates neutralizing antibodies in natural RSV infection which correlate with protection against disease. The immune response to RSV is typically short-lived, which may be related to the heavy glycosylation of RSV-G. In order to improve its immunogenicity, we expressed G protein mutants in a vesicular stomatitis virus (VSV) vector system and tested their ability to protect cotton rats from RSV challenge. We found that the most protective construct was codon-optimized RSV-G, followed by wild-type G and membrane-bound G. Constructs which expressed the G protein with reduced glycosylation or the secreted G protein provided either partial or no protection. Our results demonstrate that modifications to the G protein are not advantageous in a VSV vector system, and that an intact, codon-optimized G is a superior vaccine candidate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3' long terminal repeat (LTR) of HTLV-1. In vitro , this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8 + T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4 + T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maksimova, Smith, Seth, Phelps, Niewiesk, Satou, Green and Panfil.)