Influenza B viruses contribute substantially to seasonal disease burden; however, the structural basis by which antibodies recognize the major glycoprotein hemagglutinin (HA) and mediate antiviral activity remains incompletely defined. Influenza B virus used to circulate as two antigenically distinct lineages, B/Victoria/2/1987-like and B/Yamagata/16/1988-like, although the latter has not been detected in global surveillance in recent years. Antigenic drift in HA contributes to reduced vaccine effectiveness; however, the structural and functional basis by which antibodies recognize influenza B virus HA and mediate antiviral activity remains incompletely defined and thus thwarts our efforts in guiding next-generation vaccine design for broad protection. We characterize four murine monoclonal antibodies (mAb) that broadly bind and neutralize influenza B viruses spanning isolates across four decades of antigenic drift. Using cryo-electron microscopy coupled with in vitro and in vivo functional assays, we show that these antibodies target distinct regions of HA and confer antiviral activity through multiple mechanisms. One antibody engages the receptor-binding site and potently inhibits hemagglutination, whereas others interfere with viral egress and inhibit neuraminidase (NA) activity, suggesting steric occlusion of NA. A medial-junction antibody additionally induces antibody-dependent cellular cytotoxicity in vitro. Despite these mechanistic differences, all antibodies confer complete protection in mice when administered prophylactically or therapeutically. Together, these findings define distinct modes of antibody recognition of influenza B virus HA and link epitope specificity to antiviral function, providing a mechanistic understanding of correlates of immune protection and informing efforts to elicit broadly protective antibody responses against influenza B viruses.IMPORTANCEInfluenza B viruses cause substantial seasonal illness, particularly in children; however, antibody responses against influenza B virus remain less well understood than those against influenza A virus. Here, we identified four antibodies that broadly recognize influenza B virus hemagglutinin and protect through distinct mechanisms. We determined cryo-electron microscopy structures of three antibody-hemagglutinin complexes to define their epitopes and explain their molecular mechanisms of action. One antibody blocks viral attachment by engaging the receptor-binding site, whereas antibodies targeting the medial junction act through post-entry antiviral activity, neuraminidase inhibition, or immune effector functions. Although the antibodies differed in neutralizing potency, all protected mice when administered before infection, and several remained effective after infection. These findings show that broad protection against influenza B virus can arise through multiple antibody targets and mechanisms, informing the evaluation and design of future vaccines and antibody-based therapies.
Lone Star virus (LSV) is a bandavirus first isolated from Amblyomma americanum ticks in the United States and is phylogenetically related to severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV), and Bhanja virus, each of which has been associated with severe human disease. In contrast to these better-characterized bandaviruses, LSV remains poorly studied, and its pathogenic potential is not well defined. The recent detection of LSV RNA in the cerebrospinal fluid of an immunocompromised patient in Idaho, U.S., with fatal meningoencephalitis further highlights the need for experimental systems to investigate LSV biology. Here, we rescued recombinant LSV (rLSV) from cloned complementary DNA (cDNA) plasmids and characterized the virus. rLSV replicated similarly to the parental LSV isolate in mammalian cells and caused rapid, systemic, and lethal disease in IFNAR-/- mice, with widespread detection of viral RNA (vRNA) across multiple tissues, hepatic and splenic pathology, and induction of inflammatory cytokines. In contrast, C57BL/6J mice controlled infection and exhibited no clinical disease. To place LSV within a broader comparative framework, we generated rSFTSV from cloned cDNA and compared rLSV, rSFTSV, and HRTV in cell culture and IFNAR-/- mice. Our studies revealed distinct disease kinetics among these related tick-borne bandaviruses and showed that HRTV-induced immunity protected against homologous HRTV rechallenge and heterologous rSFTSV challenge, but not rLSV challenge. Together, these findings establish reverse-genetics platforms and small-animal models for comparative bandavirus studies, define key features of LSV pathogenesis, and place this neglected virus within a framework of related bandaviruses that differ in virulence and immunological overlap.IMPORTANCETick-borne bandaviruses include several viruses associated with severe human disease, yet many related viruses remain poorly characterized. Lone Star virus (LSV) was first isolated from Amblyomma americanum ticks decades ago, but experimental tools and animal models to study LSV infection have been lacking. Here, we generated a recombinant LSV and used it to define the outcome of infection in immunocompromised and immunocompetent mice. We show that LSV can cause rapid, systemic, and lethal disease when type I interferon signaling is absent, whereas immunocompetent mice restrict infection and remain clinically normal. By comparing LSV with Heartland virus and severe fever with thrombocytopenia syndrome virus, we also show that related tick-borne bandaviruses differ in disease kinetics and immune protection. These findings provide foundational tools for studying LSV and highlight the importance of experimentally characterizing neglected tick-borne viruses before their pathogenic potential is fully understood.
Porcine epidemic diarrhea virus (PEDV), a member of the Alphacoronavirus genus, causes severe enteric disease with up to 100% mortality in suckling piglets. Iron is an essential micronutrient for the replication of various pathogens, including viruses, which hijack host iron resources to support their proliferation. SLC40A1 encodes ferroportin (FPN), the only known iron exporter in mammals and a key target for pathogens to manipulate host iron homeostasis. However, whether coronaviruses exploit this mechanism remains unknown. Here, we demonstrate that PEDV infection disrupts host iron homeostasis through the KLF15-FPN regulatory axis, resulting in suppressed FPN expression, intracellular iron accumulation, and enhanced viral replication. Mechanistically, the PEDV envelope (E) protein induces cytoplasmic aggregation of Kruppel-like factor 15 (KLF15) through its PDZ-binding motif (PBM), promoting tripartite motif-containing protein 28 (TRIM28)-dependent K63-linked ubiquitination and autophagy-lysosomal degradation of KLF15 at lysine 339 (K339), thereby suppressing KLF15-mediated transcriptional activation of SLC40A1. Our findings uncover a novel iron hijacking strategy by which PEDV remodels host iron homeostasis through the KLF15-FPN regulatory axis, providing new insights into coronavirus-host interactions and potential antiviral intervention strategies. IMPORTANCE:Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease, posing a persistent threat to global swine production. Iron serves as a critical cofactor in numerous host and viral enzymatic processes, and profoundly influences viral infection outcomes. Ferroportin (FPN) is currently the only known iron excretory protein in mammals and has emerged as a key regulator of host iron homeostasis that can be exploited by pathogens. However, whether and how coronaviruses employ host iron regulatory pathways to remodel iron homeostasis remains unclear. Here, we reveal that PEDV E protein induces intracellular iron accumulation via the TRIM28-KLF15-FPN signaling axis, uncovering a novel function of this viral protein. This study advances our understanding of PEDV pathogenesis and highlights potential opportunities for developing anti-coronavirus strategies targeting host iron regulation.
Monoclonal antibody (mAb) cocktails are increasingly used as antiviral therapeutics, but methods for evaluating mAb combination effects remain poorly standardized. To characterize interactions of recently isolated neutralizing mAbs targeting human cytomegalovirus glycoprotein B, we compared three screening approaches: checkerboard neutralization assays; DiaMOND, a high-throughput small-molecule interaction screening tool modified for mAb combinations; and EMERALD, a modified variable-ratio framework. As the most systematic approach, the checkerboard assay served as the baseline for subsequent comparisons. Most mAb combinations in checkerboard assays were classified as additive or antagonistic according to Loewe additivity-based analyses, regardless of whether the mAbs were competing or non-competing. Apparent synergy was observed primarily at saturating antibody concentrations. When adapted for mAb combinations, the DiaMOND framework produced interaction profiles that differed from checkerboard results under both concentration-based and dose-matched conditions. In contrast, EMERALD captured concentration-dependent interactions across variable mAb ratios and revealed asymmetric behavior, depending on which mAb was held constant. Together, these findings demonstrate that classifications of mAb interactions depend strongly on assay design, concentration range, and analytical framework. They further suggest that robust mAb synergy may be less common than often reported and highlight the need to evaluate antiviral mAb combinations across multiple experimental formats.IMPORTANCEMonoclonal antibody cocktails are gaining foothold as an important strategy for the prevention and treatment of viral infections. Synergistic antibody combinations are often reported, but whether they represent true synergy is unclear because classifications of interactions depend on the experimental design and analytical methods. Moreover, there is no broadly accepted methodology for evaluating interactions among antibody combinations. As a result, it remains unclear whether common approaches for screening antibody combinations accurately capture biologically relevant interactions. Here, we compared four approaches for evaluating antibody combinations to help define best experimental and analytical practices for studying antiviral antibody interactions. Our findings demonstrate that antibody interactions are highly dependent on assay design, choice of antibody concentrations, and timing of antibody exposure, suggesting that synergy and antagonism are not fixed properties of antibody pairs. These findings may be broadly applicable to studies of antiviral antibody combinations across diverse viral systems.
Gammaherpesviruses are prevalent pathogens that are associated with cancers and autoimmune diseases. All gammaherpesviruses encode a conserved protein kinase. Expression of orf36, the protein kinase encoded by murine gammaherpesvirus 68 (MHV68), supports the establishment of a latent viral reservoir in splenic B cells in vivo. Specifically, orf36 antagonizes myeloid cell-intrinsic STAT1 function to support the passage of MHV68 from myeloid cells to splenic B cells. STAT1 is a ubiquitously expressed transcription factor and a critical effector of interferon (IFN) antiviral functions. To define the extent to which the observed orf36-STAT1 antagonism reflected orf36 antagonism of the type I IFN axis, this study employed mouse models of myeloid cell-specific type I IFN receptor (IFNAR1) or interferon regulatory factor 3 (IRF-3) deficiencies. Myeloid cell-specific IFNAR1 deficiency resulted in the rescue of the attenuated splenic latent reservoir of the orf36 null MHV68, similar to that observed upon myeloid cell-specific STAT1 deficiency, refining the viral antagonism of the IFNAR1-STAT1 axis. However, and unlike that observed for IFNAR1, myeloid cell-specific deficiency of IRF-3, the transcription factor that drives type I IFN expression, failed to affect the attenuated chronic infection of the orf36 null MHV68 mutant. Unexpectedly, wild-type MHV68 reactivation in the spleen was attenuated in mice with myeloid cell-specific IRF-3 deficiency. Thus, the current study highlights a myeloid cell-specific antagonism between conserved gammaherpesvirus protein kinase and type I IFN receptor and unveils an intriguing proviral role of myeloid-specific IRF-3 expression during chronic gammaherpesvirus infection.IMPORTANCEThis study focuses on defining the mechanisms by which conserved gammaherpesvirus protein kinase facilitates establishment of chronic infection in vivo. We previously demonstrated that the viral protein kinase antagonizes STAT1 function in myeloid cells to allow gammaherpesvirus passage from myeloid cells to B cells in the spleen. Using a combination of virus and host genetics, this study demonstrates that the previously observed antagonism between viral protein kinase and STAT1 is at least in part due to antagonism of type I interferon (IFN) signaling. Despite the well-established role of IRF-3 in type I IFN expression, myeloid cell-specific IRF-3 deficiency failed to rescue attenuated chronic infection of viral kinase null gammaherpesvirus. Unexpectedly, splenic reactivation of wild-type gammaherpesvirus was decreased in mice with myeloid cell-specific IRF-3 deficiency, unveiling a proviral role of IRF-3 expressed by myeloid cells during chronic gammaherpesvirus infection.
The study examined how 35 natural immune escape mutations (IEMs) of hepatitis B virus (HBV) located in the major antigenic loop (MAL) of the envelope proteins regulate the assembly and infectivity of the natural sub-viral agent of HBV, hepatitis delta virus (HDV). All IEMs were permissive for the assembly and secretion of HDV virions. Only seven IEMs resulted in considerably decreased levels of secreted HDV virions in the context of one to three tested HBV genotypes, B, C, or D. The effects of IEMs on HDV infectivity were much more pronounced than on the assembly. Thirteen IEMs considerably inhibited the infectivity of HDV virions. All of them greatly reduced both the levels of HDV RNA genomes accumulated in the infected cells and the percentage of HDV-infected cells. Only one mutant R(169)P generated completely non-infectious HDV virions. Our analysis of the IEMs-mediated effects on the HDV life cycle therefore identified a number of critically important amino acid residues in the MAL, and aided the mechanistic understanding of the MAL functioning during the processes of HDV assembly and infectivity. The inhibition of either HDV assembly and/or infectivity is expected to suppress the viral spread and super-infection in vivo, and therefore to reduce the HDV reservoir in infected livers. Overall, the generated data showed that the anti-HBV immune response is capable of considerable down-regulation of the concomitant HDV infection by facilitating the selection for IEMs in the MAL. The study further advanced our understanding of the mechanisms of the complex HBV-HDV and HDV-host interactions.IMPORTANCEThe study demonstrated that anti-hepatitis B virus (HBV) immune response via selection for the immune escape mutations (IEMs) in HBV envelope proteins (surface antigen, HBsAg) can considerably reduce the assembly and/or infectivity of hepatitis delta virus (HDV) virions coated with HBsAg. The IEMs could down-regulate concomitant HDV infection mostly via affecting HDV infectivity, which was considerably inhibited by 13 out of 35 IEMs. The HDV spread/super-infection, which are expected to be suppressed by inhibited via HBV-specific IEMs the assembly and/or infectivity of HDV virions, should result in a reduced HDV reservoir in infected livers. The examination of the inhibition of HDV infectivity by IEMs apparently indicated that the major antigenic loop (MAL) of HBsAg could contain the residues primarily involved in viral attachment/entry and also the residues mostly regulating intracellular post-entry trafficking of HDV virions. The data advance our understanding of the MAL functioning during infection and of the indirect regulation of the HDV life cycle through the anti-HBV immune response.
Nocturne116 is a small bacterial virus with a prolate head and a flexible, non-contractile tail that infects an insect-associated strain of Lactococcus lactis. Nocturne116 has little sequence similarity to other bacteriophages and represents a diverged virus lineage that includes the Lactococcus phage Q54 and phages of the Ceduovirus genus. To better understand the molecular architecture of this group of viruses, we determined the three-dimensional structure of the Nocturne116 virion. The head of the Nocturne116 phage is composed of two types of capsid proteins: the major capsid protein, which assembles into 50 hexamers, and a dedicated vertex protein, which forms 11 pentamers. The particle is stabilized by covalent isopeptide bonds, which result in topologically interlocked capsomer rings in the cylindrical midsection, while in the hemispherical caps, the chainmail-like structure is incomplete. An end vertex of the capsid is occupied by a portal complex, which is connected to the tail via a series of three neck protein rings. The genomic DNA extends through the neck channel up to the second tail tube ring, where it encounters the tail tape measure protein. The tail shaft is built of 26 hexameric rings of the tail tube protein and is capped by an assembly of three minor structural proteins that form a dome-shaped structure at the tail tip. The tail tip lacks characteristic receptor-binding proteins and represents the simplest host-attachment device of any studied Lactococcus phage, suggestive of an unusual mechanism by which the virus recognizes and infects its bacterial host. IMPORTANCE:Viruses that infect the widely used food fermenter Lactococcus lactis can cause major problems in the food industry, but despite considerable effort, there are still gaps in the understanding of these viruses. Knowledge of their three-dimensional structure is important for understanding aspects such as particle-stabilizing mechanisms that they employ, or the host recognition mechanisms that these viruses use. The three-dimensional structure of Lactococcus phage Nocturne116 reveals a number of unusual features such as two types of capsid proteins making up the head, covalently crosslinked capsomers which, to our knowledge, are the first such observation in a prolate-shaped virus, or a minimalist tail tip which starkly contrasts with the elaborate machinery of other previously studied Lactococcus phages. Our results also shed light on the general structure of related ceduoviruses which, despite being the third-largest lactococcal virus group, have remained poorly understood from a structural perspective.
Productive human respiratory syncytial virus (RSV) cell entry requires coordinated interactions between viral proteins and host-cell factors at the plasma membrane-actin cortex interface. Branched actin networks remodel this interface, but their precise contribution to the early stages of RSV infection remains unclear. Here, we interfered with Arp2/3 complex-dependent actin filament branching by generating A549 cell lines disrupted for expression of the essential Arp2 subunit by CRISPR/Cas9. Permanent loss of Arp2 reduced the infection of the RSV long GFP reporter virus, as quantified over the first 24 h post-infection. Compromised infection efficiency in Arp2 knockout cells persisted at later time points, and also resulted in reduced syncytia formation. Arp2 re-expression restored Arp2/3 complex-dependent actin remodeling and rescued RSV infection in Arp2 KO cells. Notably, impaired RSV infection was not accompanied by obvious changes in viral host cell attachment. Moreover, photoactivated localization microscopy (PALM) studies revealed comparable receptor diffusion and clustering in cells stably expressing mEos3.2-tagged insulin-like growth factor I receptor (IGF1R). Although Arp2/3-deficient cells displayed fewer, albeit larger, macropinosomes compared with WT cells, no changes were observed for internalized RSV genome levels. In contrast, Arp2-deficient cells appeared suppressed in RSV-F mediated virus-like particle uncoating efficiency. Consequently, viral mRNA expression and the cellular type III interferon response were reduced. Together, these data reveal that Arp2/3 complex-dependent, branched actin networks contribute to the efficiency of RSV uncoating.IMPORTANCEHuman respiratory syncytial virus (RSV) is a major cause of severe respiratory disease. The infection initiates at the plasma membrane-actin cortex interface, yet the role of actin in productive RSV entry has remained unclear. Using CRISPR/Cas9 disruption of the essential Arp2/3 complex subunit Arp2 in A549 cells, we show that branched actin networks are required for efficient RSV infection. Despite actin network remodeling, photoactivated localization microscopy showed unchanged diffusion and clustering of the RSV receptor IGF1R. Although macropinocytosis was affected in Arp2-deficient cells, RSV attachment and internalization were not influenced. In contrast, a β-lactamase virus-like-particle-based assay revealed a defect in RSV-F-mediated uncoating, followed by reduced viral gene expression and a weaker type III interferon response. These findings define Arp2/3 complex-dependent branched actin networks as a host determinant of RSV-F-mediated uncoating and provide a practical approach to quantify uncoating without engineering the RSV genome.
Major histocompatibility complex class II (MHC II)-restricted antigen presentation is central to CD4+ T-cell activation and the generation of high-affinity, class-switched antibody (Ab) responses. Peptide loading of MHC II in endosomal compartments is modulated by the functional interplay of two nonclassical MHC II molecules, H2-M and H2-O. H2-M binds to MHC II and facilitates loading of MHC II with high-affinity peptides. H2-O, a structural mimic of MHC II, modulates the MHC II pathway by binding to H2-M and inhibiting its peptide-loading catalytic activity. Although H2-O has a modest effect on the overall cell-surface MHC II peptide repertoire, these changes are sufficient to drive significant immunological consequences. H2-O plays a protective role against infection with ubiquitous γ-herpesviruses, while H2-O deficiency enables the development of protective, pathogen-specific Ab responses to mouse retroviruses and Staphylococcus aureus. Here, we further investigate the role of H2-O in regulating humoral immunity to a mouse retrovirus, mouse mammary tumor virus (MMTV). Using mosaic and conditional genetic approaches, we demonstrate that H2-O deficiency, and thus enhanced H2-M-mediated peptide loading, in either B cells or dendritic cells, is sufficient to drive a robust neutralizing Ab response against MMTV. Additionally, we show that the pronounced reduction of H2-O protein in germinal center B cells is governed by a post-transcriptional mechanism, revealing a previously unappreciated mechanism of H2-O regulation during the germinal center reaction. Collectively, these findings further establish H2-O as a tunable checkpoint in the MHC II antigen presentation pathway that shapes the quality of humoral immune responses.IMPORTANCEProtective antibody responses depend on the presentation of pathogen-derived peptides by major histocompatibility complex (MHC) class II molecules to activate CD4+ helper T cells. This process is tightly regulated. H2-O functions as a negative regulator of antigen presentation by limiting the repertoire of peptides displayed by MHC class II molecules, thereby shaping the antibody response. In this study, we show that the loss of H2-O in either of two key antigen-presenting cell types-B cells or dendritic cells-is sufficient to elicit a robust protective antibody response against a mouse retrovirus. We further demonstrate that germinal center B cells downregulate H2-O through protein degradation during the immune response. Together, these findings identify H2-O as a tunable checkpoint that regulates the magnitude and quality of antibody responses and provide additional evidence that B cells are key antigen-presenting cells.
Influenza viruses continue to pose a significant threat to human and animal health. However, the limited number of licensed antivirals is increasingly compromised by drug resistance driven by high mutation rates. This highlights the urgent need for broad-spectrum therapeutics with novel mechanisms of action. Nanobodies, a new generation of antibody drugs, have great potential in the treatment of influenza virus infections. Neuraminidase (NA) mediates the sialic acid cleavage required for viral release, and its genetic drift is generally slower than that of hemagglutinin (HA) in influenza viruses, making it an attractive target for broad-spectrum antiviral and vaccine development. To construct a phage display nanobody library targeting NA, an alpaca was immunized with the NA protein of the H9N2 influenza virus. A specific nanobody, designated F4, was subsequently screened from the immune library. The nanobody was engineered into an Fc-fused nanobody, F4-Fc, which exhibited inhibitory activity against multiple influenza A and B viruses in vitro and provided robust prophylactic and therapeutic protection against influenza A and influenza B virus infections in vivo. Mechanistically, F4-Fc inhibits NA enzymatic activity and mediates antibody-dependent cellular cytotoxicity. In conclusion, F4-Fc demonstrates prophylactic and therapeutic efficacy against influenza A and B viruses, representing a promising antiviral drug candidate for influenza virus infection.IMPORTANCEInfluenza viruses seriously threaten human and animal health, and drugs are crucial for controlling influenza outbreaks. However, the limited variety of existing anti-influenza virus medicines and the high mutation rate of the virus have led to the continuous emergence of drug-resistant strains. Neuraminidase (NA) is a critical surface glycoprotein that exhibits slower antigenic drift than hemagglutinin (HA), making it an attractive target for cross-protective antiviral development. However, broadly active NA-targeting nanobodies, particularly those effective against both influenza A and B viruses, remain limited. Here, we constructed an Fc-fused F4 nanobody (F4-Fc) targeting neuraminidases from multiple influenza A and B viruses and demonstrated its antiviral efficacy in vitro and protective activity in vivo, highlighting its potential as a promising strategy for the prevention and treatment of influenza virus infection.
Human metapneumovirus (HMPV) causes severe respiratory tract infections in all cohorts, particularly in vulnerable groups such as children, older adults, and the immunocompromised. The matrix (M) protein of HMPV, like the M proteins of other members of the order Mononegavirales, is involved in virus assembly and budding. However, other functions of the HMPV M protein have yet to be elucidated. To investigate the various functions of the M protein, we used a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) antisense agent designed to block translation of the M gene during infection. Treatment with the PPMO targeted against M mRNA (PPMOM) led to dose-dependent decreases in M expression, and a 10 µM PPMOM treatment applied at 1 hour post-infection (hpi) led to a >95% reduction in M protein at 24 hpi. Reductions in M expression had a significant impact on virus production and viral titers. Fluorescence in situ hybridization (FISH) probes used to detect vRNA showed that a reduction in M led to an increase in inclusion body (IB) count per cell, but a decrease in the average IB volume per cell at 12 and 24 hpi. HMPV-infected cells treated with PPMOM exhibited lower levels of phosphoprotein (P) at the plasma membrane. Importantly, no change in viral ribonucleoprotein movement (vRNP) was observed when the M protein levels were reduced. These studies support a model where the M protein does not facilitate movement, but instead is critical for the transfer to and stable association of vRNPs with the assembly sites at the membrane.IMPORTANCEHuman metapneumovirus (HMPV) is a respiratory pathogen that can cause severe infections in humans, but there are no targeted treatment options for HMPV. To better characterize HMPV, a matrix (M) protein-targeted peptide-conjugated phosphorodiamidate morpholino oligomer (PPMOM) was designed to control M expression during infection. Inhibition of M expression resulted in infections with reduced viral filaments and less viral RNA at the cell periphery. A reduction in M protein expression did not disrupt the active transport of viral ribonucleoproteins (vRNPs) but did significantly decrease vRNP localization at membranes and increase the number of inclusion bodies within infected cells. Viral titers were significantly reduced when M protein expression was inhibited, and dose-dependent reductions in M protein directly correlated with viral particle production. This study uses a unique tool to characterize the HMPV M protein during infection.
Human cytomegalovirus (HCMV) is a β-herpesvirus that is ubiquitous in the human population. HCMV has the largest genome of the human herpesviruses and encodes an array of genes that affect pathogenesis in different cell types. Given the ability of HCMV to replicate in a range of cell types, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded by HCMV that are homologous to G protein-coupled receptors (GPCRs); these viral GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In this study, we find that deletion of all four vGPCR genes severely attenuates HCMV replication in primary human salivary gland epithelial cells and ARPE-19 retinal epithelial cells, as evidenced by decreases in viral gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of viral gene expression and virus production. We find that, similar to US28, UL33 constitutively activates Gαq signaling to high levels in epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies provide novel molecular and biochemical data regarding UL33 expression, subcellular localization, and signaling, and indicate that UL33 activity is essential for efficient HCMV replication in cells of epithelial origin. IMPORTANCE:Human cytomegalovirus (HCMV) replicates in a number of cell types and tissues in vivo, and the viral genes involved in cell type-specific replication are just beginning to be elucidated. The HCMV-encoded viral G protein-coupled receptors (vGPCRs) UL33, UL78, US27, and US28 are proving to play important roles in multiple aspects of HCMV replication, including the establishment and maintenance of latency. Here, we demonstrate that the HCMV vGPCRs and UL33, in particular, play an important role in driving lytic replication in cells of epithelial origin, including those derived from the salivary gland. This work expands on potential functions of the vGPCRs, will drive future studies to understand mechanistically how they affect tropism, and provides a new target for future therapeutics.
Our previous studies have shown that inoculation of primary endothelial cells with BKV results in a long-term persistent infection, with inoculated cultures surviving up to 60 days while producing low levels of infectious viral progeny. Unlike the permissive renal proximal tubule cells, infected endothelial cells activate innate immunity pathways in response to BKV infection. We hypothesized that the innate immune response limits BKV infection, resulting in persistence. In this report, we show that activation of innate immune pathways is not triggered by incoming virus particles but rather occurs at a late stage of the infectious cycle, when viral DNA replication, high levels of late viral transcription, and progeny virion assembly are occurring. Single-cell transcriptomic data indicate that this response varies across the infected cell population, with some cells showing little or no innate immune response, while others appear to be highly activated. Bulk RNA-seq experiments show that the addition of interferon-β to endothelial cells results in the upregulation of a subset of known interferon-stimulated genes (ISGs), as well as many other genes not known to be associated with the interferon response. Finally, we show that the addition of interferon-β blocks BKV infection, so long as it is added prior to or soon after inoculation. These results lead to a model in which BKV infection of endothelial cells results in interferon production and ISG induction that restrict subsequent rounds of infection and lead to a persistent state. IMPORTANCE:BKV infects nearly every human on Earth, and the infection persists throughout life. The cell types where the virus persists remain a mystery, as do the state of the viral genome during persistence, as well as the mechanisms that limit productive infection and maintain persistence. While BKV is known to replicate robustly in renal proximal tubule cells during disease, it is not clear whether the virus persists in these cells. The proximal tubules are located in close proximity to endothelial cells, raising the possibility that BKV persistence occurs in the endothelia, and virus particles from infected endothelia potentially seed infections in the neighboring tubular epithelia during times of cellular stress or immunosuppression. The development of cell culture systems to study the interactions between innate immunity, virus production, and persistence is needed to advance the development of anti-viral strategies.
Powassan virus (POWV) is an emerging tick-borne flavivirus that can cause neurologic disease in humans. POWV exhibits substantial genetic and phenotypic diversity, including marked variability in replication in vitro and pathogenesis in mice. However, most experimental studies have relied on historical lineage I and II strains to assess POWV pathogenesis. Here, we characterized a contemporary lineage II strain, NY.19.12, in mice and investigated the potential viral determinants of disease. NY.19.12 caused earlier onset of clinical signs and earlier detection of viral RNA (vRNA) in the spleen and brain compared to mice infected with a widely used Midwest lineage II strain (WI.97.ic). Sequencing of NY.19.12 identified three amino acid differences in the envelope, non-structural 1, and non-structural 5 proteins. These substitutions are also present in other lineage II strains currently circulating in the Northeast United States, suggesting that they are maintained in nature. To assess their contribution to pathogenesis, the three amino acid substitutions were engineered into an infectious clone and evaluated in mice. At early time points post-infection, clinical signs and viral distribution in the brain were similar between NY.19.12 and the mutant clone, suggesting that these amino acid substitutions may contribute to disease progression and neurotropism. However, NY.19.12 vRNA was detected in the spleen at significantly higher rates than both WI.97.ic and the mutant clone, indicating that factors beyond these mutations contribute to persistence in the spleen. Together, these findings highlight the complexity of POWV pathogenesis and suggest that lineage II strains exhibit variable disease phenotypes likely driven by multiple genetic determinants.IMPORTANCETick-borne flaviviruses exhibit considerable genetic and phenotypic diversity in nature, but it is unclear if this influences their transmission and pathogenesis. Defining the mechanisms of pathogenesis requires understanding how inter-lineage variation translates to phenotypic differences in viral dissemination and neuroinvasion. This study demonstrates that even closely related Powassan virus (POWV) lineage II strains display distinct disease phenotypes that are only partially attributable to nonsynonymous consensus mutations within the viral coding sequence. By highlighting the complexity of strain-dependent POWV pathogenesis and neurotropism in mice, these findings provide valuable insights into how POWV lineage II diversity may shape disease progression and severity in humans.
A phase I study was conducted in 20 healthy subjects with 20 µg adeno-associated virus 2 (AAV2)-like particles displaying L2 residues 17-36 of both human papillomavirus (HPV) type 16 and HPV31 (AAVLP-HPV) or placebo. AAVLP-HPV vaccination was well tolerated and induced a modest L2-specific serum antibody titer that neutralizes HPV16, HPV31, and HPV5 in vitro. Here, we show that upon passive transfer into naïve mice, the sera from a subset of patients vaccinated with AAVLP-HPV conferred protection against HPV16, HPV31, and HPV5, but not HPV76. HPV16-naïve patients required three AAVLP-HPV doses to develop protection, whereas previously exposed individuals achieved protection after two doses and exhibited higher L2-specific and neutralizing antibody titers. The avidity of the L2-specific antibody response was low, but broadly cross-reactive. Prior exposure to AAV2 compromised the strength and duration of the L2 neutralizing antibody response, as well as protective immunity against HPV16, suggesting original antigenic sin. AAVLP-HPV induced high AAV2 neutralizing titers after one vaccination in AAV2-exposed or three in AAV2-naive patients. Thus, prior HPV and AAV2 infections impact the interpretation of AAVLP-HPV immunogenicity.IMPORTANCEHPV is among the most prevalent sexually transmitted infections. Persistent infection with a high-risk genotype (hrHPV) can cause cervical and other anogenital, and oropharyngeal cancers. There are a dozen hrHPV genotypes, HPV16 being the dominant type in these cancers; only ≤7 are targeted by licensed HPV vaccines. Ten intermediate-risk genotypes are also found in small proportions of cervical cancers. HPV6 and HPV11, while commonly associated with benign genital warts, can be carcinogenic in humans. The HPV+ cancer burden is highest in developing countries where cervical screening and vaccination rates are low, resulting in >500,000 cervical cancer cases annually. The plethora of skin-tropic HPVs are transmitted non-sexually and generally cause benign, self-limiting infections, but a subset of betapapillomaviruses is associated with cutaneous squamous cell carcinoma in epidermodysplasia verruciformis and immunocompromised patients. A simple, low-cost vaccine preventing infection by all of these medically significant HPV types is needed.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT03929172.
Chimeric antigen receptor T cell (CAR-T cell) therapy targeting and eliminating HIV-infected cells offers a promising approach to provide people living with HIV (PLWH) with a functional cure by preventing the recurrence of viremia caused by reactivation of latent HIV-1-infected cells. We previously described a bispecific CAR-T cell targeting two highly conserved gp120 epitopes (duoCAR-T cell) with potent anti-HIV-1 activity that is currently in clinical trials. However, elevated levels of transforming growth factor β (TGF-β) present in many PLWH may hinder the activity of both infused HIV-1-specific CAR-T cells, such as duoCAR-T cells and endogenous HIV-1-specific CD8+ T cells, thereby limiting their effectiveness to achieve a functional HIV-1 cure. We hypothesized that HCW9218, a novel bifunctional immunomodulatory protein composed of TGF-βRII and IL-15/IL-15Rα, would enhance anti-HIV-1 immunity by TGF-βRII binding and neutralizing TGF-β, while IL-15/IL-15Rα would stimulate effector cells and reactivate latent HIV-1-infected cells. We used duoCAR-T cells generated from CD4+ and CD8+ T cells from people without HIV (PWoH) and PLWH donors to demonstrate the in vitro capacity of HCW9218 to block TGF-β activity, and enhance duoCAR-T cell proliferation, cytotoxicity, and anti-HIV-1 activity. HCW9218 also functioned as a latency-reversing agent, stimulating HIV-1 production by CD4+ T cells from ART-suppressed PLWH. Production of HIV-1 by HCW9218-treated CD4+ T cells from ART-suppressed PLWH donors was suppressed by co-culture with autologous duoCAR-T cells. Together, these findings highlight the potential of HCW9218 to augment T cell and CAR-T-based therapies and contribute to strategies aimed at achieving a functional cure for HIV-1.IMPORTANCEThe persistence of HIV-1 reservoirs remains the primary barrier to an HIV-1 cure because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent HIV-1-infected cells. Treatment with anti-HIV-1 duoCAR-T cells is a potential strategy to target and eliminate HIV-1-infected cells, but their activity may be impaired by the immunosuppressive environment in lymphoid tissues of people living with HIV (PLWH). Transforming growth factor β (TGF-β), a pleiotropic cytokine elevated in PLWH, is a key mediator of this immunosuppression. Here, we show that HCW9218, a bifunctional fusion protein with TGF-β-neutralizing activity and IL-15 superagonist activity, preserves duoCAR-T cell function in the presence of TGF-β and reactivates HIV-1 production by latent HIV-1-infected cells in ART-suppressed CD4+ T cells from PLWH. These findings highlight HCW9218 as a unique dual-function immunotherapy that may enhance the efficacy of duoCAR-T cells while facilitating clearance of the HIV reservoir.
The persistence of covalently closed circular DNA (cccDNA) underlies chronic hepatitis B virus (HBV) infection and remains as the major barrier to achieving a functional cure. An accurate and sensitive quantification of cccDNA is critical for evaluating therapeutic strategies. The conventional quantitative PCR (qPCR) lacks specificity to distinguish cccDNA from viral relaxed circular DNA (rcDNA) and other replicative intermediates, while nuclease pretreatment (e.g., plasmid-safe ATP-dependent nuclease, exonucleases I/III, or T5 exonuclease) may either incompletely digest rcDNA and/or risk over-digesting cccDNA. Here, we evaluated a novel patented bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR platform that employs bisulfite treatment to disrupt rcDNA complementarity, followed by qPCR with primer sets targeting the rcDNA gap region, to enable selective amplification of cccDNA. Using synthetic HBV DNA controls and DNA samples extracted from HBV stable cell line and HBV-infected cells, we demonstrate that the BSC-cccDNA qPCR assay achieves high specificity, with tolerance of up to 109 rcDNA copies and sensitivity to 10 cccDNA copies per reaction. Comparative analyses with sensitive digital PCR (dPCR), cross-gap qPCR, and Southern blot further validated the sensitivity and accuracy of the BSC-cccDNA assay. These findings establish the BSC-cccDNA qPCR assay as a reliable tool for standardized cccDNA measurement and highlight its potential for both basic research and clinical applications.IMPORTANCEAccurate quantification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is critical for understanding viral persistence and evaluating curative therapeutic strategies. However, current PCR-based assays lack sufficient specificity to distinguish cccDNA from relaxed circular DNA (rcDNA). Enzymatic pretreatment approaches to remove rcDNA introduce variability, risk incomplete digestion, and potential cccDNA loss, thereby compromising quantitative accuracy. We evaluated a novel bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR assay that chemically disrupts rcDNA complementarity, enabling highly specific cross-gap amplification without reliance on nuclease digestion. Using synthetic substrates, cell-derived HBV DNA, and infected hepatocyte models, we demonstrate that this assay tolerates up to 10⁹ rcDNA copies while detecting as few as 10 cccDNA copies, with performance validated against Southern blot and dPCR. By improving both specificity and quantitative reliability, the BSC-cccDNA platform provides a potential standardized tool for accurate cccDNA measurement in basic research and therapeutic development.
Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with cancer and multiple sclerosis. Primary infection during adolescence significantly increases the risk of gammaherpesvirus-driven disease in humans. Correspondingly, infection of adolescent mice with murine gammaherpesvirus 68 (MHV68) has served as a tractable animal model to define host factors that control chronic gammaherpesvirus infection and pathogenesis. However, a majority of humans are infected with gammaherpesviruses during early life when the nature of adaptive immune responses is altered as compared to that of older children and adults. Given that human and murine gammaherpesviruses manipulate B cell differentiation to establish the latent reservoir in adolescents, this study aims to define the host and viral parameters of chronic gammaherpesvirus infection following neonatal inoculation. Similar to adolescents, MHV68-driven germinal center responses and the establishment of a latent viral reservoir in splenic germinal center B cells of BALB/c mice were reliably observed following neonatal intranasal infection with 400 PFU of MHV68. Unexpectedly, MHV68-driven splenomegaly, a ubiquitous clinical manifestation of gammaherpesvirus infection in recently infected adolescent mice and humans, was only observed in neonatally infected BALB/c males and not females. Interestingly, C57BL/6J neonates were largely resistant to the establishment of latent splenic infection following 400 PFU intranasal inoculation. Increasing the MHV68 inoculum rescued the establishment of chronic splenic infection and MHV68-driven germinal center responses in neonatally infected C57BL/6J mice. Thus, the current study unveils the role of genetic background and early life exposure in the susceptibility to systemic gammaherpesvirus infection.IMPORTANCEPrimary gammaherpesvirus infection of adolescents is associated with increased risk of gammaherpesvirus-driven disease, such as cancer and multiple sclerosis. Therefore, the murine gammaherpesvirus 68 (MHV68) animal model has served as a tractable experimental system to define host factors involved in control of gammaherpesvirus infection and pathogenesis following adolescent infection. However, a majority of human gammaherpesvirus infections occur during early childhood, a stage of human development that is marked by unique differences in the adaptive immune responses as compared to that of adults. This study demonstrates that the genetic background plays an important role in limiting systemic gammaherpesvirus infection following neonatal viral exposure, in contrast to that historically observed in primary adolescent infection. However, when susceptible to systemic infection, the extent of MHV68-driven germinal center responses and establishment of a latent viral reservoir in germinal center B cells are largely comparable between neonatally infected mice and published observations in adolescent MHV68 infection.
Realm Monodnaviria was established for unification of viruses with small DNA genomes that encode homologous endonucleases initiating rolling circle replication or their inactivated derivatives. However, comprehensive analysis of the expanded virus sequence and structure databases revealed that neither replication nor structural modules within this realm were monophyletic. Consequently, Monodnaviria was reorganized by moving three of its four kingdoms to separate new, likely, monophyletic realms, Efunaviria, Pleomoviria, and Volvereviria, and renaming the remaining realm Monodnaviria to Floreoviria.
The incursion of Eurasian-origin genotype A6 A(H5N5) virus into North America expanded the genetic diversity among North American highly pathogenic avian influenza viruses and heightened concern about zoonotic risk. Following a fatal human infection with the A(H5N5) virus A/Washington/2148/2025, viral replication was assessed in polarized human bronchial epithelial cells, and pathogenicity, transmissibility in direct contact and respiratory droplet models, and airborne virus shedding were evaluated in ferrets to inform pandemic risk assessment. A(H5N5) displayed robust replication in Calu-3 cells at 33°C and 37°C, showing kinetics and peak titers comparable to those of contemporary genotype B3.13 and D1.1 A(H5N1) viruses. In ferrets, A(H5N5) replicated efficiently in the respiratory tract, disseminated to extrapulmonary tissues, and caused fatal disease in all inoculated animals. Airborne transmission was not observed, and infrequent, low-level detection of virus in air samples paralleled that of A(H5) viruses that are not transmissible via air in ferrets. In a direct contact model, limited transmission was detected within 4 days of exposure, with evidence of lower respiratory tract replication in contact animals. These findings indicate that the A(H5N5) virus has the capacity for robust replication in an airway epithelial cell line and can cause severe systemic infection and mortality in ferrets but has not acquired adaptations for airborne spread in mammals. Collectively, these results underscore heterogeneity among clade 2.3.4.4b A(H5Nx) viruses in North America and the need for genotype-by-genotype evaluation of newly emerged viruses to understand public health risk.IMPORTANCEThe emergence of Eurasian-origin genotype A6 highly pathogenic avian influenza A(H5N5) virus in North America has increased viral diversity and raised concerns about zoonotic and pandemic risk. In this study, we evaluated the replication kinetics, pathogenesis, and transmission of A/Washington/2148/2025 A(H5N5) virus, which was isolated from the first reported human infection with this influenza virus subtype, using polarized human bronchial epithelial cells and the ferret model. The A(H5N5) virus replicated efficiently in vitro at temperatures representative of the upper and lower respiratory tracts and caused fatal systemic disease in inoculated ferrets. Limited transmission was observed during 4 days of direct contact. Airborne virus detection was infrequent and did not result in airborne transmission. These findings show that A(H5N5) virus can replicate robustly in mammalian cells and cause severe disease but lacks adaptations supporting efficient airborne spread, informing assessment of the pandemic risk posed by genotype A6 influenza viruses.
