Small hydrophobic (SH) proteins of Pneumoviridae and Paramyxoviridae: small but mighty.

Small hydrophobic (SH) proteins are a class of viral accessory proteins expressed by many members of the negative-stranded RNA viral families Paramyxoviridae and Pneumoviridae. Identified SH proteins are type I or II transmembrane (TM) proteins with a single-pass TM domain. Little is known about the functions of SH proteins; however, several possess viroporin activity, enhancing membrane permeability of infected cells or those expressing SH protein. Moreover, several SH proteins inhibit apoptosis and immune signaling pathways within infected cells, including TNF and interferon signaling, or activate inflammasomes. SH proteins are generally nonessential for viral replication in vitro, but loss of SH is often associated with reduced replication in vivo, suggesting a role in enhancing viral replication or evading host immunity. Analogous proteins are expressed by a variety of pathogens of public health importance; thus, understanding the functional importance and mechanisms of SH proteins provides insight into the pathogenesis and replication of negative-sense RNA viruses.

Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

Cleavage of the syncytial protein of J paramyxovirus is required for its ability to promote cell-cell fusion.

Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the "LKTG" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.

Culturing Immortalized Human Airway Epithelial Cells at an Air-Liquid Interface for Measles Virus Infection.

Measles virus (MeV) infection of airway surface epithelial cells provides a site for final amplification before being released back into the environment via coughing and sneezing. Multiple cell lines have served as models of polarized epithelia for MeV infection, such as Caco2 cells (intestinal derived human epithelia) or MDCK cells (kidney derived canine epithelia). In this chapter, we describe the materials and air-liquid interface (ALI) culture conditions for maintaining four different cell lines derived from human airway epithelial cells: 16HBE14o-, Calu-3, H358, and NuLi-1. We provide methods for confirming transepithelial electrical resistance (TER) and preparing samples for microscopy as well as expected results from apical or basolateral MeV delivery. Polarized human airway derived cells serve as tissue culture models for investigating targeted questions about how MeV exits a human host. In addition, these methods are generalizable to studies of other respiratory viruses or the biology of ALI airway epithelial cells.

Phase I/II Trial of Urokinase Plasminogen Activator-Targeted Oncolytic Newcastle Disease Virus for Canine Intracranial Tumors.

Neurotropic oncolytic viruses are appealing agents to treat brain tumors as they penetrate the blood-brain barrier and induce preferential cytolysis of neoplastic cells. The pathobiological similarities between human and canine brain tumors make immunocompetent dogs with naturally occurring tumors attractive models for the study of oncolytic virotherapies. In this dose-escalation/expansion study, an engineered Lasota NDV strain targeting the urokinase plasminogen activator system (rLAS-uPA) was administered by repetitive intravenous infusions to 20 dogs with intracranial tumors with the objectives of characterizing toxicities, immunologic responses, and neuroradiological anti-tumor effects of the virus for up to 6 months following treatment. Dose-limiting toxicities manifested as fever, hematologic, and neurological adverse events, and the maximum tolerated dose (MTD) of rLAS-uPA was 2 × 107 pfu/mL. Mild adverse events, including transient infusion reactions, diarrhea, and fever were observed in 16/18 of dogs treated at or below MTD. No infectious virus was recoverable from body fluids. Neutralizing antibodies to rLAS-uPA were present in all dogs by 2 weeks post-treatment, and viral genetic material was detected in post-treatment tumors from six dogs. Tumor volumetric reductions occurred in 2/11 dogs receiving the MTD. Systemically administered rLAS-uPA NDV was safe and induced anti-tumor effects in canine brain tumors, although modifications to evade host anti-viral immunity are needed to optimize this novel therapy.

Prefusion stabilization of the Hendra and Langya virus F proteins.

Nipah virus (NiV) and Hendra virus (HeV) are pathogenic paramyxoviruses that cause mild-to-severe disease in humans. As members of the Henipavirus genus, NiV and HeV use an attachment (G) glycoprotein and a class I fusion (F) glycoprotein to invade host cells. The F protein rearranges from a metastable prefusion form to an extended postfusion form to facilitate host cell entry. Prefusion NiV F elicits higher neutralizing antibody titers than postfusion NiV F, indicating that stabilization of prefusion F may aid vaccine development. A combination of amino acid substitutions (L104C/I114C, L172F, and S191P) is known to stabilize NiV F in its prefusion conformation, although the extent to which substitutions transfer to other henipavirus F proteins is not known. Here, we perform biophysical and structural studies to investigate the mechanism of prefusion stabilization in F proteins from three henipaviruses: NiV, HeV, and Langya virus (LayV). Three known stabilizing substitutions from NiV F transfer to HeV F and exert similar structural and functional effects. One engineered disulfide bond, located near the fusion peptide, is sufficient to stabilize the prefusion conformations of both HeV F and LayV F. Although LayV F shares low overall sequence identity with NiV F and HeV F, the region around the fusion peptide exhibits high sequence conservation across all henipaviruses. Our findings indicate that substitutions targeting this site of conformational change might be applicable to prefusion stabilization of other henipavirus F proteins and support the use of NiV as a prototypical pathogen for henipavirus vaccine antigen design.IMPORTANCEPathogenic henipaviruses such as Nipah virus (NiV) and Hendra virus (HeV) cause respiratory symptoms, with severe cases resulting in encephalitis, seizures, and coma. The work described here shows that the NiV and HeV fusion (F) proteins share common structural features with the F protein from an emerging henipavirus, Langya virus (LayV). Sequence alignment alone was sufficient to predict which known prefusion-stabilizing amino acid substitutions from NiV F would stabilize the prefusion conformations of HeV F and LayV F. This work also reveals an unexpected oligomeric interface shared by prefusion HeV F and NiV F. Together, these advances lay a foundation for future antigen design targeting henipavirus F proteins. In this way, Nipah virus can serve as a prototypical pathogen for the development of protective vaccines and monoclonal antibodies to prepare for potential henipavirus outbreaks.

Upper and/or Lower Respiratory Tract Infection Caused by Human Metapneumovirus After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Human metapneumovirus (hMPV) epidemiology, clinical characteristics and risk factors for poor outcome after allogeneic stem cell transplantation (allo-HCT) remain a poorly investigated area. METHODS: This retrospective multicenter cohort study examined the epidemiology, clinical characteristics, and risk factors for poor outcomes associated with human metapneumovirus (hMPV) infections in recipients of allo-HCT. RESULTS: We included 428 allo-HCT recipients who developed 438 hMPV infection episodes between January 2012 and January 2019. Most recipients were adults (93%). hMPV infections were diagnosed at a median of 373 days after allo-HCT. The infections were categorized as upper respiratory tract disease (URTD) or lower respiratory tract disease (LRTD), with 60% and 40% of cases, respectively. Patients with hMPV LRTD experienced the infection earlier in the transplant course and had higher rates of lymphopenia, neutropenia, corticosteroid use, and ribavirin therapy. Multivariate analysis identified lymphopenia and corticosteroid use (>30 mg/d) as independent risk factors for LRTD occurrence. The overall mortality at day 30 after hMPV detection was 2% for URTD, 12% for possible LRTD, and 21% for proven LRTD. Lymphopenia was the only independent risk factor associated with day 30 mortality in LRTD cases. CONCLUSIONS: These findings highlight the significance of lymphopenia and corticosteroid use in the development and severity of hMPV infections after allo-HCT, with lymphopenia being a predictor of higher mortality in LRTD cases.

Immune responses elicited by ssRNA(-) oncolytic viruses in the host and in the tumor microenvironment.

Oncolytic viruses (OVs) are at the forefront of biologicals for cancer treatment. They represent a diverse landscape of naturally occurring viral strains and genetically modified viruses that, either as single agents or as part of combination therapies, are being evaluated in preclinical and clinical settings. As the field gains momentum, the research on OVs has been shifting efforts to expand our understanding of the complex interplay between the virus, the tumor and the immune system, with the aim of rationally designing more efficient therapeutic interventions. Nowadays, the potential of an OV platform is no longer defined exclusively by the targeted replication and cancer cell killing capacities of the virus, but by its contribution as an immunostimulator, triggering the transformation of the immunosuppressive tumor microenvironment (TME) into a place where innate and adaptive immunity players can efficiently engage and lead the development of tumor-specific long-term memory responses. Here we review the immune mechanisms and host responses induced by ssRNA(-) (negative-sense single-stranded RNA) viruses as OV platforms. We focus on two ssRNA(-) OV candidates: Newcastle disease virus (NDV), an avian paramyxovirus with one of the longest histories of utilization as an OV, and influenza A (IAV) virus, a well-characterized human pathogen with extraordinary immunostimulatory capacities that is steadily advancing as an OV candidate through the development of recombinant IAV attenuated platforms.

Interferon-stimulated gene TDRD7 interacts with AMPK and inhibits its activation to suppress viral replication and pathogenesis.

IMPORTANCE: Virus infection triggers induction of interferon (IFN)-stimulated genes (ISGs), which ironically inhibit viruses themselves. We identified Tudor domain-containing 7 (TDRD7) as a novel antiviral ISG, which inhibits viral replication by interfering with autophagy pathway. Here, we present a molecular basis for autophagy inhibitory function of TDRD7. TDRD7 interacted with adenosine monophosphate (AMP)-activated protein kinase (AMPK), the kinase that initiates autophagy, to inhibit its activation. We identified domains required for the interaction; deleting AMPK-interacting domain blocked antiAMPK and antiviral activities of TDRD7. We used primary cells and mice to evaluate the TDRD7-AMPK antiviral pathway. TDRD7-deficient primary mouse cells exhibited enhanced AMPK activation and viral replication. Finally, TDRD7 knockout mice showed increased susceptibility to respiratory virus infection. Therefore, our study revealed a new antiviral pathway of IFN and its contribution to host response. Our results have therapeutic potential; a TDRD7-derived peptide may be an effective AMPK inhibitor with application as antiviral agent.

Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein.

IMPORTANCE: Genetically diverse paramyxoviruses are united in their presentation of a receptor-binding protein (RBP), which works in concert with the fusion protein to facilitate host-cell entry. The C-terminal head region of the paramyxoviral RBP, a primary determinant of host-cell tropism and inter-species transmission potential, forms structurally distinct classes dependent upon protein and glycan receptor specificity. Here, we reveal the architecture of the C-terminal head region of the RBPs from Nariva virus (NarV) and Mossman virus (MosV), two archetypal rodent-borne paramyxoviruses within the recently established genus Narmovirus, family Paramyxoviridae. Our analysis reveals that while narmoviruses retain the general architectural features associated with paramyxoviral RBPs, namely, a six-bladed ß-propeller fold, they lack the structural motifs associated with known receptor-mediated host-cell entry pathways. This investigation indicates that the RBPs of narmoviruses exhibit pathobiological features that are distinct from those of other paramyxoviruses.

Structures of Langya Virus Fusion Protein Ectodomain in Pre- and Postfusion Conformation.

Langya virus (LayV) is a paramyxovirus in the Henipavirus genus, closely related to the deadly Nipah (NiV) and Hendra (HeV) viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-electron microscopy (cryo-EM) structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and postfusion conformations. The LayV-F protein exhibits pre- and postfusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and postfusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide (FP) is buried within a highly conserved, hydrophobic interprotomer pocket in the prefusion state and is notably less flexible than the rest of the protein, highlighting its "spring-loaded" state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to postfusion conversion that may apply more broadly to paramyxoviruses. IMPORTANCE The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which have important vaccine and therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to the Paramyxoviridae family.

The HRA2pl fusion peptide exerts in vitro antiviral activity against human respiratory paramyxoviruses and pneumoviruses.

Acute respiratory infections are a group of diseases caused by viruses, bacteria, and parasites that mainly affect children until the age of 5 and immunocompromised senior adults. In Mexico, these infections are the main cause of morbidity in children, with more than 26 million cases of respiratory infections reported by the Secretariat of Health, in 2019. The human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV), and the human parainfluenza-2 (hPIV-2) are responsible for many respiratory infections. Currently, palivizumab, a monoclonal antibody against the fusion protein F, is the treatment of choice against hRSV infections. This protein is being studied for the design of antiviral peptides that act by inhibiting the fusion of the virus and the host cell. Therefore, we examined the antiviral activity of the HRA2pl peptide, which competes the heptad repeat A domain of the F protein of hMPV. The recombinant peptide was obtained using a viral transient expression system. The effect of the fusion peptide was evaluated with an in vitro entry assay. Moreover, the effectiveness of HRA2pl was examined in viral isolates from clinical samples obtained from patients with infections caused by hRSV, hMPV, or hPIV-2, by evaluating the viral titer and the syncytium size. The HRA2pl peptide affected the viruses' capacity of entry, resulting in a 4-log decrease in the viral titer compared to the untreated viral strains. Additionally, a 50% reduction in the size of the syncytium was found. These results demonstrate the antiviral potential of HRA2pl in clinical samples, paving the way toward clinical trials.

Avian Paramyxovirus 4 Antitumor Activity Leads to Complete Remissions and Long-term Protective Memory in Preclinical Melanoma and Colon Carcinoma Models.

Avulaviruses represent a diverse subfamily of non-segmented negative strand RNA viruses infecting avian species worldwide. To date, 22 different serotypes have been identified in a variety of avian hosts, including wild and domestic birds. APMV-1, also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized due to its relevance for the poultry industry and, more recently, its inherent oncolytic activity and potential as a cancer therapeutic. An array of both naturally-occurring and recombinant APMV-1 strains has been tested in different preclinical models and clinical trials, highlighting NDV as a promising viral agent for human cancer therapy. To date, the oncolytic potential of other closely related avulaviruses remains unknown. Here, we have examined the in vivo anti-tumor capability of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8 and -9 in syngeneic murine colon carcinoma and melanoma tumor models. Our studies have identified APMV-4 Duck/Hong Kong/D3/1975 virus as a novel oncolytic agent with greater therapeutic potential than one of the NDV clinical candidate strains, La Sota. Intratumoral administration of the naturally-occurring APMV-4 virus significantly extends survival, promotes complete remission, and confers protection against re-challenge in both murine colon carcinoma and melanoma tumor models. Furthermore, we have designed a plasmid rescue strategy that allows us to develop recombinant APMV-4-based viruses. The infectious clone rAPMV-4 preserves the extraordinary antitumor capacity of its natural counterpart, paving the way to a promising next generation of viral therapeutics.

Ribavirin for Treatment of Subjects with Respiratory Syncytial Virus-Related Infection: A Systematic Review and Meta-Analysis.

INTRODUCTION: Respiratory syncytial virus (RSV)-associated diseases have caused an estimated 1.8 million hospital admissions and 40,000 deaths among children. RSV can cause lower respiratory tract infections (LRTIs) in all age groups, adults with comorbidities, and immunocompromised patients. The aim was to summarize the evidence concerning efficacy and safety of ribavirin in subjects diagnosed with RSV associated with LRTI. METHODS: A systematic review and meta-analysis were performed. Eligible studies were observational (> 10 subjects) and randomized-controlled trials of subjects with aerosol/oral ribavirin for RSV-LRTI. Comparator was supportive care or placebo. Systematic search on PubMed, Cochrane Library, and Web of Science databases was conducted between January 2001 and January 2022. PROSPERO register number: CRD42022308147. RESULTS: After retrieving 907 studies, 10 observational studies and 1 randomized controlled trial were included (4/11 high quality of evidence). Seven studies included subjects with haematological malignancy/stem cell transplant, two lung transplants, and two healthy individuals. A total of 788 subjects diagnosed with RSV infection were included; 14.3% of them presented with only LRTI. Among 445 subjects treated with ribavirin, 195 (43.8%) received an aerosolized formulation. Pooled meta-analysis showed no differences in mortality [risk ratio (RR): 0.63; 95% confidence interval (CI): 0.28-1.42] in all subjects treated with aerosol/oral ribavirin compared to supportive care. In subgroup analysis, mortality was significantly lower in haematological subjects (RR: 0.32; 95% CI: 0.14-0.71), but did not differ significantly in lung transplant recipients (RR: 0.89; 95% CI 0.31-2.56). Oral ribavirin (vs. supportive care) was associated with increased viral clearance (RR: 2.60; 95% CI: 1.35-4.99). Seventeen adverse events were reported among 119 subjects, but none were severe. CONCLUSION: Ribavirin should be considered for treatment of RSV-LRTI in haematological subjects. There is a lack of evidence to support its use in lung transplant recipients. Oral formulation appears to be an easier, safe, and cost-effective alternative to aerosolized ribavirin. Further advances needs to focus on newer antivirals.

A molecular, epidemiological and pathogenicity analysis of pigeon paramyxovirus type 1 viruses isolated from live bird markets in China in 2014-2021.

To expand our understanding of the epidemiology of pigeon paramyxovirus type 1 (PPMV-1) in China, risk-based active surveillance was undertaken with pigeon swabs collected from live bird markets in 2014-2021. Seventy-six PPMV-1 strains were isolated from 12 provinces (60%) of the 20 provinces surveyed, and the positive rates of PPMV-1 varied from 0.50% to 3.19% annually. The complete genomic sequences of 18 representative viruses were analyzed, revealing a genome of 15,192 nucleotides, with the gene order 3'-NP-P-M-F-HN-L-5'. All isolates contained the 112RRQKRF117 cleavage site in the fusion (F) protein, a characteristic generally associated with virulent Newcastle disease viruses (NDVs), and the intracerebral pathogenicity index values (1.05-1.41) of four isolates indicated their virulence. A challenge experiment also demonstrated that all four isolates are pathogenic to pigeons, with morbidity rates of 60-100% and mortality rates of 0-30%. A further analysis of the functional domains of the F and HN proteins revealed several mutations in the fusion peptide, signal peptide, neutralizing epitopes, heptad repeat region, and transmembrane domains, and the substitution of cysteine residue 25 (C25Y) and substitutions in the HRb region (V287I) of the F protein and the transmembrane domain (V45A) of the HN protein may play important roles in PPMV-1 virulence. In a phylogenetic analysis based on the complete sequences of the F gene, all eighteen isolates all clustered into sub-genotype VI.2.1.1.2.2 (VIb) in class II, and shared high nucleotide sequence identity, indicating that the PPMV-1 strains in sub-genotype VI.2.1.1.2.2 are the predominant PPMV-1 viruses in pigeons in China and that the variations in these viruses have been relatively stable over the past 8 years. This study identifies the genetic and pathogenicity characteristics of the PPMV-1 strains prevalent in China and extends our understanding of the prevalence of this virus in China.

Novel Roles of the Nipah Virus Attachment Glycoprotein and Its Mobility in Early and Late Membrane Fusion Steps.

The Paramyxoviridae family comprises important pathogens that include measles (MeV), mumps, parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). Paramyxoviral entry into cells requires viral-cell membrane fusion, and formation of paramyxoviral pathognomonic syncytia requires cell-cell membrane fusion. Both events are coordinated by intricate interactions between the tetrameric attachment (G/H/HN) and trimeric fusion (F) glycoproteins. We report that receptor binding induces conformational changes in NiV G that expose its stalk domain, which triggers F through a cascade from prefusion to prehairpin intermediate (PHI) to postfusion conformations, executing membrane fusion. To decipher how the NiV G stalk may trigger F, we introduced cysteines along the G stalk to increase tetrameric strength and restrict stalk mobility. While most point mutants displayed near-wild-type levels of cell surface expression and receptor binding, most yielded increased NiV G oligomeric strength, and showed remarkably strong defects in syncytium formation. Furthermore, most of these mutants displayed stronger F/G interactions and significant defects in their ability to trigger F, indicating that NiV G stalk mobility is key to proper F triggering via moderate G/F interactions. Also remarkably, a mutant capable of triggering F and of fusion pore formation yielded little syncytium formation, implicating G or G/F interactions in a late step occurring post fusion pore formation, such as the extensive fusion pore expansion required for syncytium formation. This study uncovers novel mechanisms by which the G stalk and its oligomerization/mobility affect G/F interactions, the triggering of F, and a late fusion pore expansion step-exciting novel findings for paramyxoviral attachment glycoproteins. IMPORTANCE The important Paramyxoviridae family includes measles, mumps, human parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). The deadly emerging NiV can cause neurologic and respiratory symptoms in humans with a >60% mortality rate. NiV has two surface proteins, the receptor binding protein (G) and fusion (F) glycoproteins. They mediate the required membrane fusion during viral entry into host cells and during syncytium formation, a hallmark of paramyxoviral and NiV infections. We previously discovered that the G stalk domain is important for triggering F (via largely unknown mechanisms) to induce membrane fusion. Here, we uncovered new roles and mechanisms by which the G stalk and its mobility modulate the triggering of F and also unexpectedly affect a very late step in membrane fusion, namely fusion pore expansion. Importantly, these novel findings may extend to other paramyxoviruses, offering new potential targets for therapeutic interventions.

Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524).

The necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn. IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

Headless Henipaviral Receptor Binding Glycoproteins Reveal Fusion Modulation by the Head/Stalk Interface and Post-receptor Binding Contributions of the Head Domain.

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we demonstrate quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct C-terminal stalk lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3- to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Novel Paju Apodemus paramyxovirus 1 and 2, harbored by Apodemus agrarius in the Republic of Korea.

Paramyxoviruses harbored by multiple natural reservoirs pose a potential threat to public health. Jeilongvirus has been proposed as a novel paramyxovirus genus found in rodents, bats, and cats. Paramyxovirus RNA was detected in 108/824 (13.1%) Apodemus agrarius captured at 14 trapping sites in the Republic of Korea. We first present two genetically distinct novel paramyxoviruses, Paju Apodemus paramyxovirus 1 (PAPV-1) and 2 (PAPV-2). The disparity between PAPV-1 (19,716 nucleotides) and -2 (17,475 nucleotides) revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogeny of PAPV-1 and -2 belonged to distinct genetic lineages of Jeilongvirus, respectively, even though these viruses were originated from A. agrarius. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and pro-inflammatory cytokines. Therefore, this study provides insights into the molecular epidemiology, genetic diversity, and virus-host interactions of novel rodent-borne paramyxoviruses.