Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Structural insight into arenavirus replication machinery.

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.

Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.

CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.

Cell entry by SARS-CoV-2.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) invades host cells by interacting with receptors/coreceptors, as well as with other cofactors, via its spike (S) protein that further mediates fusion between viral and cellular membranes. The host membrane protein, angiotensin-converting enzyme 2 (ACE2), is the major receptor for SARS-CoV-2 and is a crucial determinant for cross-species transmission. In addition, some auxiliary receptors and cofactors are also involved that expand the host/tissue tropism of SARS-CoV-2. After receptor engagement, specific proteases are required that cleave the S protein and trigger its fusogenic activity. Here we discuss the recent advances in understanding the molecular events during SARS-CoV-2 entry which will contribute to developing vaccines and therapeutics.

African swine fever virus A137R assembles into a dodecahedron cage.

African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications. IMPORTANCE: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity.

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic-type serine-threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG-host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin-activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-ß-activated kinase 1 (TAK1), thereby inhibiting the activation of NF-κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), rather than the usual C86-Ub thiol-ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.

Molecular basis of Coxsackievirus A10 entry using the two-in-one attachment and uncoating receptor KRM1.

KREMEN1 (KRM1) has been identified as a functional receptor for Coxsackievirus A10 (CV-A10), a causative agent of hand-foot-and-mouth disease (HFMD), which poses a great threat to infants globally. However, the underlying mechanisms for the viral entry process are not well understood. Here we determined the atomic structures of different forms of CV-A10 viral particles and its complex with KRM1 in both neutral and acidic conditions. These structures reveal that KRM1 selectively binds to the mature viral particle above the canyon of the viral protein 1 (VP1) subunit and contacts across two adjacent asymmetry units. The key residues for receptor binding are conserved among most KRM1-dependent enteroviruses, suggesting a uniform mechanism for receptor binding. Moreover, the binding of KRM1 induces the release of pocket factor, a process accelerated under acidic conditions. Further biochemical studies confirmed that receptor binding at acidic pH enabled CV-A10 virion uncoating in vitro. Taken together, these findings provide high-resolution snapshots of CV-A10 entry and identify KRM1 as a two-in-one receptor for enterovirus infection.

Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4.

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

Molecular Basis of Binding between Middle East Respiratory Syndrome Coronavirus and CD26 from Seven Bat Species.

Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting humans have occurred since the identification of this virus in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozen spillover infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with equilibrium dissociation constant values ranging from several to hundreds at the micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.IMPORTANCE In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.

Molecular Basis of a Protective/Neutralizing Monoclonal Antibody Targeting Envelope Proteins of both Tick-Borne Encephalitis Virus and Louping Ill Virus.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development.IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

Structures of human-infecting Thogotovirus fusogens support a common ancestor with insect baculovirus.

Thogotoviruses are emerging tick-borne zoonotic orthomyxoviruses infecting both humans and domestic animals with severe clinical consequences. These viruses utilize a single-envelope glycoprotein (Gp) to facilitate their entry into host cells. Here, we present the Gp structures of Thogoto and Dhori viruses, both of which are members of the Thogotovirus genus in the family Orthomyxoviridae These structures, determined in the postfusion conformation, identified them as class III viral fusion proteins. It is intriguing that the Gp structures are similar to the envelope protein of baculovirus, although sharing a low sequence identity of ∼28%. Detailed structural and phylogenic analyses demonstrated that these Gps originated from a common ancestor. Among the structures, domain I is the most conserved region, particularly the fusion loops. Domain II showed the highest variability among different viruses, which might be related to their distinct host tropism. These findings increase our understanding of the divergent evolution processes of various orthomyxoviruses and indicate potential targets for developing antiviral therapeutics by intercepting virus entry.

Postfusion structure of human-infecting Bourbon virus envelope glycoprotein.

Thogotoviruses are important zoonotic viruses infecting a variety of domestic animals, as well as humans. Among these viruses, Bourbon virus (BRBV) is one of the several human-infecting members, which emerged in the US in recent years and caused human deaths. Here, we report the crystal structure of the BRBV envelope glycoprotein in the postfusion conformation. The structure adopts the typical fold of a class III viral fusion protein and displays an extensive positively charged electrostatic potential pattern, which resembles the glycoprotein of Dhori virus and is consistent with our previous predictions. In addition, compared to other previously defined class III viral fusion proteins, the structures of all thogotovirus glycoproteins and homologs are more similar to herpes virus glycoprotein Bs than to the rhabdovirus G proteins. Thus, class III viral fusion proteins are quite diverse in structure, and sub-classes may have developed during evolution.

The Postfusion Structure of the Heartland Virus Gc Glycoprotein Supports Taxonomic Separation of the Bunyaviral Families Phenuiviridae and Hantaviridae.

Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low-pH conditions. Here, we describe the crystal structure of HRTV Gc in its postfusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct features in the aspects of subdomain orientation, N-linked glycosylation, the interaction pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens.IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee on Taxonomy of Viruses reclassification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two ß-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

Glycan Binding Specificity and Mechanism of Human and Porcine P[6]/P[19] Rotavirus VP8*s.

Rotaviruses (RVs), which cause severe gastroenteritis in infants and children, recognize glycan ligands in a genotype-dependent manner via the distal VP8* head of the spike protein VP4. However, the glycan binding mechanisms remain elusive for the P[II] genogroup RVs, including the widely prevalent human RVs (P[8], P[4], and P[6]) and a rare P[19] RV. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s and found that the P[II] genogroup RV VP8*s could commonly interact with mucin core 2, which may play an important role in RV evolution and cross-species transmission. We determined the first P[6] VP8* structure, as well as the complex structures of human P[19] VP8*, with core 2 and lacto-N-tetraose (LNT). A glycan binding site was identified in human P[19] VP8*. Structural superimposition and sequence alignment revealed the conservation of the glycan binding site in the P[II] genogroup RV VP8*s. Our data provide significant insight into the glycan binding specificity and glycan binding mechanism of the P[II] genogroup RV VP8*s, which could help in understanding RV evolution, transmission, and epidemiology and in vaccine development.IMPORTANCE Rotaviruses (RVs), belonging to the family Reoviridae, are double-stranded RNA viruses that cause acute gastroenteritis in children and animals worldwide. Depending on the phylogeny of the VP8* sequences, P[6] and P[19] RVs are grouped into genogroup II, together with P[4] and P[8], which are widely prevalent in humans. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s, determined the crystal structure of P[6] VP8*, and uncovered the glycan binding pattern in P[19] VP8*, revealing a conserved glycan binding site in the VP8*s of P[II] genogroup RVs by structural superimposition and sequence alignment. Our data suggested that mucin core 2 may play an important role in P[II] RV evolution and cross-species transmission. These data provide insight into the cell attachment, infection, epidemiology, and evolution of P[II] genogroup RVs, which could help in developing control and prevention strategies against RVs.

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs.IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention.

Functional and Structural Characterization of P[19] Rotavirus VP8* Interaction with Histo-blood Group Antigens.

Rotaviruses (RVs) of species A (RVA) are a major causative agent of acute gastroenteritis. Recently, histo-blood group antigens (HBGAs) have been reported to interact with human RVA VP8* proteins. Human P[19] is a rare P genotype of porcine origin that infects humans sporadically. The functional and structural characteristics of P[19] VP8* interaction with HBGAs are unknown. In this study, we expressed and purified the VP8* proteins of human and porcine P[19] RVs. In oligosaccharide and saliva binding assays, P[19] VP8* proteins showed obvious binding to A-, B-, and O-type saliva samples irrespective of the secretor status, implying broad binding patterns. However, they did not display specific binding to any of the oligosaccharides tested. In addition, we solved the structure of human P[19] VP8* at 2.4 Å, which revealed a typical galectin-like fold. The structural alignment demonstrated that P[19] VP8* was most similar to that of P[8], which was consistent with the phylogenetic analysis. Structure superimposition revealed the basis for the lack of binding to the oligosaccharides. Our study indicates that P[19] RVs may bind to other oligosaccharides or ligands and may have the potential to spread widely among humans. Thus, it is necessary to place the prevalence and evolution of P[19] RVs under surveillance. IMPORTANCE: Human P[19] is a rare P genotype of porcine origin. Based on phylogenetic analysis of VP8* sequences, P[19] was classified in the P[II] genogroup, together with P[4], P[6], and P[8], which have been reported to interact with HBGAs in a genotype-dependent manner. In this study, we explored the functional and structural characteristics of P[19] VP8* interaction with HBGAs. P[19] VP8* showed binding to A-, B-, and O-type saliva samples, as well as saliva of nonsecretors. This implies that P[19] has the potential to spread among humans with a broad binding range. Careful attention should be paid to the evolution and prevalence of P[19] RVs. Furthermore, we solved the structure of P[19] VP8*. Structure superimposition indicated that P[19] may bind to other oligosaccharides or ligands using potential binding sites, suggesting that further investigation of the specific cell attachment factors is warranted.

The Wag31 protein interacts with AccA3 and coordinates cell wall lipid permeability and lipophilic drug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis, especially drug resistant tuberculosis, is a serious threat to global human health. Compared with other bacterial pathogens, M. tuberculosis gains stronger natural drug resistance from its unusually lipid-rich cell wall. As a DivIVA homolog, Wag31 has been demonstrated to be closely involved in peptidoglycan synthesis, cell growth and cell division. Previous research rarely investigated the role of Wag31 in drug resistance. In this study, we found Wag31 knock-down in Mycobacterium smegmatis resulted in a co-decrease of the resistance to four lipophilic drugs (rifampicin, novobiocin, erythromycin and clofazimine) and an increase in the cell permeability to lipophilic molecules. Six proteins (AccA3, AccD4 and AccD5, Fas, InhA and MmpL3) that are involved in fatty acid and mycolic acid synthesis were identified in the Wag31 interactome through Co-Immunoprecipitation. The Wag31-AccA3 interaction was confirmed by the pull-down assay. AccA3 overexpression resulted in a decrease in lipid permeability and an increase in the resistance of rifampicin and novobiocin. It confirmed the close relationship of lipophilic drug resistance, lipid permeability and the Wag31-AccA3 interaction. These results demonstrated that Wag31 maintained the resistance to lipophilic drugs and that Wag31 could play a role in controlling the lipid permeability of the cell wall through the Wag31-AccA3 interaction.