Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

A potent Henipavirus cross-neutralizing antibody reveals a dynamic fusion-triggering pattern of the G-tetramer.

The Hendra and Nipah viruses (HNVs) are highly pathogenic pathogens without approved interventions for human use. In addition, the interaction pattern between the attachment (G) and fusion (F) glycoproteins required for virus entry remains unclear. Here, we isolate a panel of Macaca-derived G-specific antibodies that cross-neutralize HNVs via multiple mechanisms. The most potent antibody, 1E5, confers adequate protection against the Nipah virus challenge in female hamsters. Crystallography demonstrates that 1E5 has a highly similar binding pattern to the receptor. In cryo-electron microscopy studies, the tendency of 1E5 to bind to the upper or lower heads results in two distinct quaternary structures of G. Furthermore, we identify the extended outer loop ß1S2-ß1S3 of G and two pockets on the apical region of fusion (F) glycoprotein as the essential sites for G-F interactions. This work highlights promising drug candidates against HNVs and contributes deeper insights into the viruses.

Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L.

The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host's endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication.

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

A Bacterium-like Particle Vaccine Displaying Envelope Proteins of Canine Distemper Virus Can Induce Immune Responses in Mice and Dogs.

Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F3-GEM and H4-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F3 and H4-GEM was also designed, a mixture consisting of F3-GEM and H4-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F3-GEM, H4-GEM or F3 and H4-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F3 and H4-GEM group was higher than the other two groups. Additionally, F3 and H4-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F3 and H4-GEM induce IgG and neutralizing antibodies' response in dogs. Conclusions: In summary, F3 and H4-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F3 and H4-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

In silico design of a novel multi-epitope vaccine against HCV infection through immunoinformatics approaches.

Infection with the hepatitis C virus (HCV) is one of the causes of liver cancer, which is the world's sixth most prevalent and third most lethal cancer. The current treatments do not prevent reinfection; because they are expensive, their usage is limited to developed nations. Therefore, a prophylactic vaccine is essential to control this virus. Hence, in this study, an immunoinformatics method was applied to design a multi-epitope vaccine against HCV. The best B- and T-cell epitopes from conserved regions of the E2 protein of seven HCV genotypes were joined with the appropriate linkers to design a multi-epitope vaccine. In addition, cholera enterotoxin subunit B (CtxB) was included as an adjuvant in the vaccine construct. This study is the first to present this epitopes-adjuvant combination. The vaccine had acceptable physicochemical characteristics. The vaccine's 3D structure was predicted and validated. The vaccine's binding stability with Toll-like receptor 2 (TLR2) and TLR4 was confirmed using molecular docking and molecular dynamics (MD) simulation. The immune simulation revealed the vaccine's efficacy by increasing the population of B and T cells in response to vaccination. In silico expression in Escherichia coli (E. coli) was also successful.

Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly.

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance.

The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.