The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus.

Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern, with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue virus (DENV), a flavivirus closely related to ZIKV. Here we investigated the serological cross-reaction between the two viruses. Plasma immune to DENV showed substantial cross-reaction to ZIKV and was able to drive antibody-dependent enhancement (ADE) of ZIKV infection. Using a panel of human monoclonal antibodies (mAbs) to DENV, we showed that most antibodies that reacted to DENV envelope protein also reacted to ZIKV. Antibodies to linear epitopes, including the immunodominant fusion-loop epitope, were able to bind ZIKV but were unable to neutralize the virus and instead promoted ADE. Our data indicate that immunity to DENV might drive greater ZIKV replication and have clear implications for disease pathogenesis and future vaccine programs for ZIKV and DENV.

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus.

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2.

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

Extensive flavivirus E trimer breathing accompanies stem zippering of the post-fusion hairpin.

Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein trimers from the tick-borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process.

Structural basis of potent Zika-dengue virus antibody cross-neutralization.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Overexpression of UBA5 in Cells Mimics the Phenotype of Cells Lacking UBA5.

Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5's active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination-a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer.

Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

Antibody response in elderly vaccinated four times with an mRNA anti-COVID-19 vaccine.

The humoral response after the fourth dose of a mRNA vaccine against COVID-19 has not been adequately described in elderly recipients, particularly those not exposed previously to SARS-CoV-2. Serum anti-RBD IgG levels (Abbott SARS-CoV-2 IgG II Quant assay) and neutralizing capacities (spike SARS-CoV-2 pseudovirus Wuhan and Omicron BA.1 variant) were measured after the third and fourth doses of a COVID-19 mRNA vaccine among 46 elderly residents (median age 85 years [IQR 81; 89]) of an assisted living facility. Among participants never infected by SARS-CoV-2, the mean serum IgG levels against RBD (2025 BAU/ml), 99 days after the fourth vaccine, was as high as 76 days after the third vaccine (1987 BAU/ml), and significantly higher (p = 0.030) when the latter were corrected for elapsed time. Neutralizing antibody levels against the historical Wuhan strain were significantly higher (Mean 1046 vs 1573; p = 0.002) and broader (against Omicron) (Mean 170 vs 375; p = 0.018), following the fourth vaccine. The six individuals with an Omicron breakthrough infection mounted strong immune responses for anti-RBD and neutralizing antibodies against the Omicron variant indicating that the fourth vaccine dose did not prevent a specific adaptation of the immune response. These findings point out the value of continued vaccine boosting in the elderly population.

Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment.

Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(ß-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.

Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery.

The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH reveals the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged pr-binding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation.

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces neutralizing salivary IgA.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

RNA polymerase III and antiviral innate immune response.

The innate immune system has numerous signal transduction pathways that lead to the production of type I interferons in response to exposure of cells to external stimuli. One of these pathways comprises RNA polymerase (Pol) III that senses common DNA viruses, such as cytomegalovirus, vaccinia, herpes simplex virus-1 and varicella zoster virus. This polymerase detects and transcribes viral genomic regions to generate AU-rich transcripts that bring to the induction of type I interferons. Remarkably, Pol III is also stimulated by foreign non-viral DNAs and expression of one of its subunits is induced by an RNA virus, the Sindbis virus. Moreover, a protein subunit of RNase P, which is known to associate with Pol III in initiation complexes, is induced by viral infection. Accordingly, alliance of the two tRNA enzymes in innate immunity merits a consideration.

Caveolin 2 regulates endocytosis and trafficking of the M1 muscarinic receptor in MDCK epithelial cells.

Clathrin and caveolins are known for their involvement in the internalization of numerous receptors. Here we show that in polarized epithelial Madin-Darby canine kidney cells, both the clathrin machinery and caveolins are involved in the endocytosis and delivery to the plasma membrane (PM) of the M1 muscarinic acetylcholine receptor (mAChR). We initially localized this receptor to the lateral membrane, where it accumulates proximal to the tight junctions. From there it is internalized through the clathrin-mediated pathway. In addition, the receptor may associate on the PM with caveolin (cav) 2 or in intracellular compartments with either cav 2, or monomeric or oligomeric cav 1. Association of the PM M1 mAChR with cav 2 inhibits receptor endocytosis through the clathrin-mediated pathway or retains the receptor in an intracellular compartment. This intracellular association attenuates receptor trafficking. Expression of cav 1 with cav 2 rescues the latter's inhibitory effect. The caveolins stimulate M1 mAChR oligomerization thus maintaining a constant amount of monomeric receptor. These results provide evidence that caveolins play a role in the attenuation of the M1 muscarinic receptor's intracellular trafficking to and from the PM.

Glycan-Glycan Interaction Determines Shigella Tropism toward Human T Lymphocytes.

Direct interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis.IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells.

Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope.

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic 'breathing' of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs.

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into ß-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.