Expression, purification, and crystal structure of mpox virus A41 protein.

Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus A41L gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (KD) being 1.22 × 10-6 M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic ß-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.

Cryo-EM Structure of human HCN3 channel and its regulation by cAMP.

HCN channels are important for regulating heart rhythm and nerve activity and have been studied as potential drug targets for treating depression, arrhythmia, nerve pain and epilepsy.Despite possessing unique pharmacological properties, HCN channels share common characteristics in that they are activated by hyperpolarization and modulated by cAMP and other membrane lipids. However, the mechanisms of how these ligands bind and modulate HCN channels are unclear. In this study, we solved structures of full-length human HCN3 using cryo-EM and captured two different states, including a state without any ligand bound and a state with cAMP bound. Our structures reveal the novel binding sites for cholesteryl hemisuccinate in apo-state and show how cholesteryl hemisuccinate and cAMP binding cause conformational changes in different states. These findings explain how these small modulators are sensed in mammals at the molecular level.The results of our study could help design more potent and specific compounds to influence HCN channel activity and offer new therapeutic possibilities for diseases that lack effective treatment.

Structural Basis for Coronaviral Main Proteases Inhibition by the 3CLpro Inhibitor GC376.

The main protease (Mpro) of coronaviruses participates in viral replication, serving as a hot target for drug design. GC376 is able to effectively inhibit the activity of Mpro, which is due to nucleophilic addition of GC376 by binding covalently with Cys145 in Mpro active site. Here, we used fluorescence resonance energy transfer (FRET) assay to analyze the IC50 values of GC376 against Mpros from six different coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-HUK1, MERS-CoV, SARS-CoV, HCoV-NL63) and five Mpro mutants (G15S, M49I, K90R, P132H, S46F) from SARS-CoV-2 variants. The results showed that GC376 displays effective inhibition to various coronaviral Mpros and SARS-CoV-2 Mpro mutants. In addition, the crystal structures of SARS-CoV-2 Mpro (wide type)-GC376, SARS-CoV Mpro-GC376, MERS-CoV Mpro-GC376, and SARS-CoV-2 Mpro mutants (G15S, M49I, S46F, K90R, and P132H)-GC376 complexes were solved. We found that GC376 is able to fit into the active site of Mpros from different coronaviruses and different SARS-CoV-2 variants properly. Detailed structural analysis revealed key molecular determinants necessary for inhibition and illustrated the binding patterns of GC376 to these different Mpros. In conclusion, we not only proved the inhibitory activity of GC376 against different Mpros including SARS-CoV-2 Mpro mutants, but also revealed the molecular mechanism of inhibition by GC376, which will provide scientific guidance for the development of broad-spectrum drugs against SARS-CoV-2 as well as other coronaviruses.

Crystal structure of SARS-CoV-2 main protease (Mpro) mutants in complex with the non-covalent inhibitor CCF0058981.

SARS-CoV-2 constantly circulates and evolves worldwide, generating many variants and posing a menace to global health. It is urgently needed to discover effective medicines to treat the disease caused by SARS-CoV-2 and its variants. An established target for anti-SARS-CoV-2 drug discovery is the main protease (Mpro), since it exerts an irreplaceable action in viral life cycle. CCF0058981, derived from ML300, is a non-covalent inhibitor that exhibits low nanomolar potency against SARS-CoV-2 Mpro and submicromolar anti-SARS-CoV-2 activity, thereby providing a valuable starting point for drug design. However, structural basis underlying inhibition of SARS-CoV-2 Mpro by CCF0058981 remains undetermined. In this study, the crystal structures of CCF0058981 in complex with two SARS-CoV-2 Mpro mutants (M49I and V186F), which have been identified in the recently emerged Omicron subvariants, were solved. Structural analysis defined the pivotal molecular factors responsible for the interactions between CCF0058981 and these two Mpro mutants, and revealed the binding modes of CCF0058981 to Mpro M49I and V186F mutants. These data not only provide structural insights for SARS-CoV-2 Mpro inhibition by CCF0058981, but also add to develop effective broad-spectrum drugs against SARS-CoV-2 as well as its variants.

A potent neutralizing nanobody targeting a unique epitope on the receptor-binding domain of SARS-CoV-2 spike protein.

SARS-CoV-2 and its variants continue to threaten public health. Nanobodies that block the attachment of the RBD to host cell angiotensin-converting enzyme 2 (ACE2) represent promising drug candidates. In this study, we reported the identification and structural biological characterization of a nanobody from a RBD-immunized alpaca. The nanobody, termed as 2S-1-19, shows outstanding neutralizing activity against both pseudotyped and authentic SARS-CoV-2 viruses. The crystal structure of 2S-1-19 bound to SARS-CoV-2 RBD reveals an epitope that overlaps with the binding site for ACE2. We also showed that 2S-1-19 reserves promising, though compromised, neutralizing activity against the Delta variant and that the trivalent form of 2S-1-19 remarkably increases its neutralizing capacity. Despite this, neither the monomeric or trimeric 2S-1-19 could neutralize the Omicron BA.1.1 variant, possibility due to the E484A and Q493K mutations found within this virus variant. These data provide insights into immune evasion caused by SARS-CoV-2 variants.

Crystal structures of main protease (Mpro) mutants of SARS-CoV-2 variants bound to PF-07304814.

There is an urgent need to develop effective antiviral drugs to prevent the viral infection caused by constantly circulating SARS-CoV-2 as well as its variants. The main protease (Mpro) of SARS-CoV-2 is a salient enzyme that plays a vital role in viral replication and serves as a fascinating therapeutic target. PF-07304814 is a covalent inhibitor targeting SARS-CoV-2 Mpro with favorable inhibition potency and drug-like properties, thus making it a promising drug candidate for the treatment of COVID-19. We previously solved the structure of PF-07304814 in complex with SARS-CoV-2 Mpro. However, the binding modes of PF-07304814 with Mpros from evolving SARS-CoV-2 variants is under-determined. In the current study, we expressed six Mpro mutants (G15S, K90R, M49I, S46F, V186F, and Y54C) that have been identified in Omicron variants including the recently emerged XBB.1.16 subvariant and solved the crystal structures of PF-07304814 bound to Mpro mutants. Structural analysis provided insight into the key molecular determinants responsible for the interaction between PF-07304814 and these mutant Mpros. Patterns for PF-07304814 to bind with these investigated Mpro mutants and the wild-type Mpro are generally similar but with some differences as revealed by detailed structural comparison. Structural insights presented in this study will inform the development of novel drugs against SARS-CoV-2 and the possible conformation changes of Mpro mutants when bound to an inhibitor.

Structural basis for the inhibition of coronaviral main proteases by ensitrelvir.

Main protease (Mpro) is a highly conserved cysteine protease that plays a vital role in the replication of coronaviruses, making it an attractive pan-coronaviral therapeutic target. Ensitrelvir (S-217622), developed by Shionogi, is the first orally active non-covalent, non-peptidic SARS-CoV-2 Mpro inhibitor, which also displays antiviral efficacy against other human coronaviruses as well as SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). Here, we report the crystal structures of the main proteases from SARS-CoV-2, SARS-CoV-2 VOC/VOIs, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor S-217622. A detailed analysis of these structures illuminates key structural determinants essential for inhibition and elucidates the binding modes of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of coronaviral infection, structural insights obtained from this study could accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.

Crystal structures of main proteases of SARS-CoV-2 variants bound to a benzothiazole-based inhibitor.

Main protease (M pro) serves as an indispensable factor in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as its constantly emerging variants and is therefore considered an attractive target for antiviral drug development. Benzothiazole-based inhibitors targeting M pro have recently been investigated by several groups and proven to be promising leads for coronaviral drug development. In the present study, we determine the crystal structures of a benzothiazole-based inhibitor, YH-53, bound to M pro mutants from SARS-CoV-2 variants of concern (VOCs) or variants of interest (VOIs), including K90R (Beta, B.1.351), G15S (Lambda, C.37), Y54C (Delta, AY.4), M49I (Omicron, BA.5) and P132H (Omicron, B.1.1.529). The structures show that the benzothiazole group in YH-53 forms a C-S covalent bond with the sulfur atom of catalytic residue Cys145 in SARS-CoV-2 M pro mutants. Structural analysis reveals the key molecular determinants necessary for interaction and illustrates the binding mode of YH-53 to these mutant M pros. In conclusion, structural insights from this study offer more information to develop benzothiazole-based drugs that are broader spectrum, more effective and safer.

Evaluation of the Inhibition Potency of Nirmatrelvir against Main Protease Mutants of SARS-CoV-2 Variants.

SARS-CoV-2 continues to pose a threat to public health. Main protease (Mpro) is one of the most lucrative drug targets for developing specific antivirals against SARS-CoV-2 infection. By targeting Mpro, peptidomimetic nirmatrelvir is able to inhibit viral replication of SARS-CoV-2 and reduce the risk for progression to severe COVID-19. However, multiple mutations in the gene encoding Mpro of emerging SARS-CoV-2 variants raise a concern of drug resistance. In the present study, we expressed 16 previously reported SARS-CoV-2 Mpro mutants (G15S, T25I, T45I, S46F, S46P, D48N, M49I, L50F, L89F, K90R, P132H, N142S, V186F, R188K, T190I, and A191V). We evaluated the inhibition potency of nirmatrelvir against these Mpro mutants and solved the crystal structures of representative Mpro mutants of SARS-CoV-2 bound to nirmatrelvir. Enzymatic inhibition assays revealed that these Mpro variants remain susceptible to nirmatrelvir as the wildtype. Detailed analysis and structural comparison provided the inhibition mechanism of Mpro mutants by nirmatrelvir. These results informed the ongoing genomic surveillance of drug resistance of emerging SARS-CoV-2 variants to nirmatrelvir and facilitate the development of next-generation anticoronavirus drugs.

Structural basis for a human broadly neutralizing influenza A hemagglutinin stem-specific antibody including H17/18 subtypes.

Influenza infection continues are a persistent threat to public health. The identification and characterization of human broadly neutralizing antibodies can facilitate the development of antibody drugs and the design of universal influenza vaccines. Here, we present structural information for the human antibody PN-SIA28's heterosubtypic binding of hemagglutinin (HA) from circulating and emerging potential influenza A viruses (IAVs). Aside from group 1 and 2 conventional IAV HAs, PN-SIA28 also inhibits membrane fusion mediated by bat-origin H17 and H18 HAs. Crystallographic analyses of Fab alone or in complex with H1, H14, and H18 HA proteins reveal that PN-SIA28 binds to a highly conserved epitope in the fusion domain of different HAs, with the same CDRHs but different CDRLs for different HAs tested, distinguishing it from other structurally characterized anti-stem antibodies. The binding characteristics of PN-SIA28 provides information to support the design of increasingly potent engineered antibodies, antiviral drugs, and/or universal influenza vaccines.

Structural Basis for the Inhibition of Coronaviral Main Proteases by a Benzothiazole-Based Inhibitor.

The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV Mpros. Crystal structures of Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of Mpros from other coronaviruses. In consideration of the important role of Mpro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral Mpro inhibitors that are safer and more effective.

Structural Basis of Main Proteases of Coronavirus Bound to Drug Candidate PF-07304814.

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.

Structural Basis of the Main Proteases of Coronavirus Bound to Drug Candidate PF-07321332.

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.

Potential Inhibitors Targeting Papain-Like Protease of SARS-CoV-2: Two Birds With One Stone.

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), the pathogen of the Coronavirus disease-19 (COVID-19), is still devastating the world causing significant chaos to the international community and posing a significant threat to global health. Since the first outbreak in late 2019, several lines of intervention have been developed to prevent the spread of this virus. Nowadays, some vaccines have been approved and extensively administered. However, the fact that SARS-CoV-2 rapidly mutates makes the efficacy and safety of this approach constantly under debate. Therefore, antivirals are still needed to combat the infection of SARS-CoV-2. Papain-like protease (PLpro) of SARS-CoV-2 supports viral reproduction and suppresses the innate immune response of the host, which makes PLpro an attractive pharmaceutical target. Inhibition of PLpro could not only prevent viral replication but also restore the antiviral immunity of the host, resulting in the speedy recovery of the patient. In this review, we describe structural and functional features on PLpro of SARS-CoV-2 and the latest development in searching for PLpro inhibitors. Currently available inhibitors targeting PLpro as well as their structural basis are also summarized.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Crystal structures of human coronavirus NL63 main protease at different pH values.

Human coronavirus NL63 (HCoV-NL63), which belongs to the genus Alphacoronavirus, mainly infects children and the immunocompromized and is responsible for a series of clinical manifestations, including cough, fever, rhinorrhoea, bronchiolitis and croup. HCoV-NL63, which was first isolated from a seven-month-old child in 2004, has led to infections worldwide and accounts for 10% of all respiratory illnesses caused by etiological agents. However, effective antivirals against HCoV-NL63 infection are currently unavailable. The HCoV-NL63 main protease (Mpro), also called 3C-like protease (3CLpro), plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins (pp1a and pp1ab) into functional subunits. Moreover, Mpro is highly conserved among all coronaviruses, thus making it a prominent drug target for antiviral therapy. Here, four crystal structures of HCoV-NL63 Mpro in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with Mpro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in different genera and extends knowledge of the diversity, function and evolution of coronaviruses.

Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus.

Here, we investigate a monoclonal antibody, Z2B3, isolated from an H7N9-infected patient, that exhibited cross-reactivity to both N9 (group 2) and a broad range of seasonal and avian N1 (group 1) proteins but lost activity to the N1 with the substitution K432E. This substitution exists in 99.25% of seasonal influenza strains after 2013. The NA-Z2B3 complex structures indicated that Z2B3 binds within the conserved active site of the neuraminidase (NA) protein. A salt bridge between D102 in Z2B3 and K432 in NA plays an important role in binding. Structure-based modification of Z2B3 with D102R in heavy chain reversed the salt bridge and restored the binding and inhibition of N1 with E432. Furthermore, Z2B3-D102R can protect mice from A/Serbia/NS-601/2014 H1N1 virus (NA contains E432) infection while the wild-type Z2B3 antibody shows no protection. This study demonstrates that a broadly reactive and protective antibody to NA can be in principle edited to restore binding and inhibition to recently drifted N1 NA and regain protection against the variant influenza strain.IMPORTANCE The immune system produces antibodies to protect the human body from harmful invaders. The monoclonal antibody (MAb) is one kind of effective antivirals. In this study, we isolated an antibody (Z2B3) from an H7N9 influenza virus-infected child. It shows cross-reactivity to both group 1 (N1) and group 2 (N9) neuraminidases (NAs) but is sensitive to N1 NA with a K432E substitution. Structural analysis of the NA-antibody fragment antigen-binding (Fab) complex provides a clue for antibody modification, and the modified antibody restored binding and inhibition to recently drifted N1 NA and regained protection against the variant influenza strain. This finding suggests that antibodies to NA may be a useful therapy and can be in principle edited to defeat drifted influenza virus.

Neutralization mechanism of human monoclonal antibodies against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

Structure-function analysis of neutralizing antibodies to H7N9 influenza from naturally infected humans.

Little is known about the specificities and neutralization breadth of the H7-reactive antibody repertoire induced by natural H7N9 infection in humans. We have isolated and characterized 73 H7-reactive monoclonal antibodies from peripheral B cells from four donors infected in 2013 and 2014. Of these, 45 antibodies were H7-specific, and 17 of these neutralized the virus, albeit with few somatic mutations in their variable domain sequences. An additional set of 28 antibodies, isolated from younger donors born after 1968, cross-reacted between H7 and H3 haemagglutinins in binding assays, and had accumulated significantly more somatic mutations, but were predominantly non-neutralizing in vitro. Crystal structures of three neutralizing and protective antibodies in complex with the H7 haemagglutinin revealed that they recognize overlapping residues surrounding the receptor-binding site of haemagglutinin. One of the antibodies, L4A-14, bound into the sialic acid binding site and made contacts with haemagglutinin residues that were conserved in the great majority of 2016-2017 H7N9 isolates. However, only 3 of the 17 neutralizing antibodies retained activity for the Yangtze River Delta lineage viruses isolated in 2016-2017 that have undergone antigenic change, which emphasizes the need for updated H7N9 vaccines.

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site-Specific Glycan Structures.

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.