NEDD4 family ubiquitin ligases associate with LCMV Z's PPXY domain and are required for virus budding, but not via direct ubiquitination of Z.

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli-derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence.

Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required ∼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.

Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly.

We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories. These viral RNA structures are highly dynamic during acute infection, with the many small foci seen early coalescing into larger perinuclear foci later in infection. These late-forming perinuclear viral RNA aggregates are located near the cellular microtubule organizing centre and colocalize with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA and viral glycoprotein, prior to their transport to the plasma membrane, where new particles will bud.

A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles.

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

Endogenous distal airway progenitor cells, lung mechanics, and disproportionate lobar growth following long-term postpneumonectomy in mice.

Using a model of postpneumonectomy (PNY) compensatory lung growth in mice, we previously observed an increase in numbers of a putative endogenous distal airway progenitor cell population (CCSP(pos) /pro-SPC(pos) cells located at bronchoalveolar duct junctions [BADJs]), at 3, 7, and 14 days after pneumonectomy, returning to baseline at 28 days post-PNY. As the origin of these cells is poorly understood, we evaluated whether bone marrow cells contributed to the pool of these or other cells during prolonged post-PNY lung regrowth. Naïve and sex-mismatched chimeric mice underwent left PNY and were evaluated at 1, 2, and 3 months for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells and presence of donor-derived marrow cells engrafted as airway or alveolar epithelium. Nonchimeric mice were also examined at 12 months after PNY for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells. Notably, the right accessory lobe (RAL) continued to grow disproportionately over 12 months, a novel finding not previously described. Assessment of lung mechanics demonstrated an increase in lung stiffness following PNY, which significantly diminished over 1 year, but remained elevated relative to 1-year-old naïve controls. However, the number of CCSP(pos) /pro-SPC(pos) BADJ cells ≥1-month following PNY was equivalent to that found in naïve controls even after 12 months of continued RAL growth. Notably, no donor bone marrow-derived cells engrafted as airway or alveolar epithelial cells, including those at the BADJ, up to 3 months after PNY. These studies suggest that lung epithelial cells, including CCSP(pos) /pro-SPC(pos) cells, are not replenished from marrow-derived cells during post-PNY lung growth in mice.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice.

Bone marrow-derived mesenchymal stromal cells (BMSCs) mitigate inflammation in mouse models of acute lung injury. However, specific mechanisms of BMSC actions on CD4 T lymphocyte-mediated inflammation in vivo remain poorly understood. Limited data suggests promotion of Th2 phenotype in models of Th1-mediated diseases. However, whether this might alleviate or worsen Th2-mediated diseases such as allergic asthma is unknown. To ascertain the effects of systemic administration of BMSCs in a mouse model of Th2-mediated allergic airways inflammation, ovalbumin (OVA)-induced allergic airways inflammation was induced in wild-type C57BL/6 and BALB/c mice as well as in interferon-γ (IFNγ) receptor null mice. Effects of systemic administration during antigen sensitization of either syngeneic or allogeneic BMSC on airways hyperreactivity, lung inflammation, antigen-specific CD4 T lymphocytes, and serum immunoglobulins were assessed. Both syngeneic and allogeneic BMSCs inhibited airways hyperreactivity and lung inflammation through a mechanism partly dependent on IFNγ. However, contrary to existing data, BMSCs did not affect antigen-specific CD4 T lymphocyte proliferation but rather promoted Th1 phenotype in vivo as assessed by both OVA-specific CD4 T lymphocyte cytokine production and OVA-specific circulating immunoglobulins. BMSCs treated to prevent release of soluble mediators and a control cell population of primary dermal skin fibroblasts only partly mimicked the BMSC effects and in some cases worsened inflammation. In conclusion, BMSCs inhibit Th2-mediated allergic airways inflammation by influencing antigen-specific CD4 T lymphocyte differentiation. Promotion of a Th1 phenotype in antigen-specific CD4 T lymphocytes by BMSCs is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNγ-dependent process.

Comparison of VSV Pseudovirus and Focus Reduction Neutralization Assays for Measurement of Anti-Andes orthohantavirus Neutralizing Antibodies in Patient Samples.

Andes orthohantavirus (ANDV) is the etiologic agent of hantavirus cardiopulmonary syndrome (HCPS), which has a case fatality rate around 35%, with no effective treatment or vaccine available. ANDV neutralizing antibody (NAb) measurements are important for the evaluation of the immune response following infection, vaccination, or passive administration of investigational monoclonal or polyclonal antibodies. The standard assay for NAb measurement is a focus reduction neutralization test (FRNT) featuring live ANDV and must be completed under biosafety level (BSL)-3 conditions. In this study, we compared neutralization assays featuring infectious ANDV or vesicular stomatitis virus (VSV) pseudovirions decorated with ANDV glycoproteins for their ability to measure anti-ANDV NAbs from patient samples. Our studies demonstrate that VSV pseudovirions effectively measure NAb from clinical samples and have greater sensitivity compared to FRNT with live ANDV. Importantly, the pseudovirus assay requires less labor and sample materials and can be conducted at BSL-2.

Stimulation of B Cell Immunity in Flavivirus-Naive Individuals by the Tetravalent Live Attenuated Dengue Vaccine TV003.

The tetravalent live attenuated dengue vaccine candidate TV003 induces neutralizing antibodies against all four dengue virus serotypes (DENV1-DENV4) and protects against experimental challenge with DENV2 in humans. Here, we track vaccine viremia and B and T cell responses to this vaccination/challenge model to understand how vaccine viremia links adaptive immunity and development of protective antibody responses. TV003 viremia triggers an acute plasmablast response that, in combination with DENV-specific CD4+ T cells, correlates with serum neutralizing antibodies. TV003 vaccinees develop DENV2-reactive memory B cells, including serotype-specific and multivalent specificities in line with the composition of serum antibodies. There is no post-challenge plasmablast response in vaccinees, although stronger and earlier post-TV003 plasmablast responses associate with sterile humoral protection from DENV2 challenge. TV003 vaccine triggers plasmablasts and memory B cells, which, with support from CD4+ T cells, functionally link early vaccine viremia and the serum antibody responses.

Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells.

RATIONALE: Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases. OBJECTIVES: We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium. METHODS: Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells. MEASUREMENTS AND MAIN RESULTS: When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. CONCLUSIONS: CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.

Host-Driven Phosphorylation Appears to Regulate the Budding Activity of the Lassa Virus Matrix Protein.

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function.

The intracellular cargo receptor ERGIC-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles.

Arenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.