Yeping Sun

Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus

Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence analysis of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, analysis at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of soluble hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.

The membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic T-lymphocyte epitopes

Abstract Background. Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged with highly contagious and life-threatening characteristics in 2002, remains a potential risk for future outbreaks. Membrane (M) and envelope (E) proteins are major structural proteins of the SARS-CoV. The M protein has been determined as a protective antigen in humoral responses. However, its potential roles in stimulating cellular immunity remain elusive. Methods. In this study, a panel of peptides derived from M and E proteins were tested by in vitro refolding, T2 cell-binding assays, and responses stimulated by cytotoxic T-lymphocyte (CTL) epitopes in HLA-A2.1/Kb transgenic mice and human peripheral blood mononuclear cells (PBMCs). Results. A nonameric epitope Mn2 and a decameric epitope Md3 derived from the M protein were identified and used for the evaluation of M protein-specific immunity. Responses stimulated by M protein-specific CTL epitopes have been found in the PBMCs of donors who had recovered from SARS infection. Additionally, the transmembrane domain of the M protein may contain a T cell epitope cluster revealed by the immunogenic and structural analysis of a panel of truncated peptides overlapping with Mn2 and Md3. Conclusions. The M protein of SARS-CoV holds dominant cellular immunogenicity. This, together with previous reports of a strong humoral response against the M protein, may help to further explain the immunogenicity of SARS and serves as potential targets for SARS-CoV vaccine design.

In silico characterization of the functional and structural modules of the hemagglutinin protein from the swine-origin influenza virus A (H1N1)-2009.

The 2009 swine-origin influenza virus (S-OIV, H1N1 subtype) has developed into a new pandemic influenza as announced by the World Health Organization. In order to uncover clues about the determinants for virulence and pathogenicity of the virus, we characterized the functional modules of the surface glycoprotein hemagglutinin (HA), the most important protein in molecular epidemiology and pathogenesis of influenza viruses. We analyzed receptor binding sites, basic patch, neutralization antibody epitopes and T cell epitopes in the HA protein of the current S-OIV according to the corresponding functional and structural modules previously characterized in other H1 HA molecules or HA molecules of other subtypes. We compared their differences and similarities systematically. Based on the amino acids defined as the functional and structural modules, the HA protein of 2009 S-OIV should specifically bind to the human 2,6-receptor. The D225G/E mutation in HA, which is found in some isolates, may confer dual binding specificity to the 2,3- and 2,6-receptor based on previously reported work. This HA variant contains two basic patches, one of which results in increased basicity, suggesting enhanced membrane fusion function. The 2009 S-OIV HA also has an extra glycosylation site at position 276. Four of the five antibody neutralization epitopes identified in A/RP/8/34(H1N1) were exposed, but the other was hidden by a glycosylation site. The previously identified cytotoxic T cell epitopes in various HA molecules were summarized and their corresponding sequences in 2009 S-OIV HA were defined. These results are critical for understanding the pathogenicity of the virus and host immune response against the virus.

Identification and structural definition of H5-specific CTL epitopes restricted by HLA-A*0201 derived from the H5N1 subtype of influenza A viruses

The haemagglutinin (HA) glycoprotein of influenza A virus is a major antigen that initiates humoral immunity against infection; however, the cellular immune response against HA is poorly understood. Furthermore, HA-derived cytotoxic T-lymphocyte (CTL) epitopes are relatively rare in comparison to other internal gene products. Here, CTL epitopes of the HA serotype H5 protein were screened. By using in silico prediction, in vitro refolding and a T2 cell-binding assay, followed by immunization of HLA-A2.1/K(b) transgenic mice, an HLA-A*0201-restricted decameric epitope, RI-10 (H5 HA205-214, RLYQNPTTYI), was shown to elicit a robust CTL epitope-specific response. In addition, RI-10 and its variant, KI-10 (KLYQNPTTYI), were also demonstrated to be able to induce a higher CTL epitope-specific response than the influenza A virus dominant CTL epitope GL-9 (GILGFVFTL) in peripheral blood mononuclear cells of HLA-A*0201-positive patients who had recovered from H5N1 virus infection. Furthermore, the crystal structures of RI-10-HLA-A*0201 and KI-10-HLA-A*0201 complexes were determined at 2.3 and 2.2 A resolution, respectively, showing typical HLA-A*0201-restricted epitopes. The conformations of RI-10 and KI-10 in the antigen-presenting grooves in crystal structures of the two complexes show significant differences, despite their nearly identical sequences. These results provide implications for the discovery of diagnostic markers and the design of novel influenza vaccines.

It is not just AIV: From avian to swine-origin influenza virus

In March and early April 2009, a new swine-origin influ-enza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. The virus spreads worldwide by human-to-human transmission. Within a few weeks, it reached a pandemic level. The virus is a novel reassorment virus. It contains gene fragments of influenza virus of swine, avian and human emerged from a triple reassortant virus circulating in North American swine.

Conserved epitopes dominate cross‐CD8+ T‐cell responses against influenza A H1N1 virus among Asian populations

Novel strains of influenza A viruses (IAVs) have emerged with high infectivity and/or pathogenicity in recent years, causing worldwide concern. T cells are correlated with protection in humans through cross‐reactive immunity against heterosubtypes of IAV. However, the different hierarchical roles of IAV‐derived epitopes with distinct levels of polymorphism in the cross‐reactive T‐cell responses against IAV remain elusive. In this study, immunodominant epitopes scattered throughout the entire proteome of 2009 pandemic influenza A H1N1 virus and seasonal IAVs were synthesized and divided into different pools depending on their conservation. The overall profile of the IAV‐specific CD8+ T‐cell immunity was detected by utilizing these peptide pools and also individual peptides. A dominant role of the conserved peptide‐specific T‐cell immunity was illuminated within the anti‐IAV responses, while the CD8+ T‐cell responses against the variable epitopes were lower than the conserved peptides. As previously demonstrated within a Caucasian population, we determined that GL9‐specific T cells, which also utilize Vβ 17 TCR (BV19), play a pivotal role in IAV‐specific T‐cell immunity within an HLA‐A2+ Asian population. Our study objectively reveals the different predominant roles of T‐cell epitopes among IAV‐specific cross‐reactive cellular immunity. This may guide the development of vaccines with cross‐T‐cell immunogenicity against heterosubtypes of IAV.

From endocytosis to membrane fusion: emerging roles of dynamin in virus entry

Dynamin, a large guanosine triphosphatase (GTPase), has been implicated in virus entry, but its mechanisms of action are controversial. The entry procedure of most enveloped viruses involves endocytosis and membrane fusion. Dynamin has been suggested to act both as a regulatory GTPase by controlling the early stages of clathrin-mediated endocytosis (CME), which is an important endocytic pathway utilized by many viruses, and as a mechanchemical enzyme that induces membrane fission and pinches endocytic vesicles off from the cellular plasma membrane in later stages in several endocytic pathways, including CME. In addition to its involvement in virus endocytosis, dynamin has also been proposed to participate in membrane fusion between the virus and endosomes following endocytosis. Crystal structures and cryo-electron micrography (cryo-EM) have elucidated the structure of dynamin, which led to development of a mechanochemical model of how dynamin-mediated membrane fission occurs. Based on this, we propose a hypothetical model that explains how dynamin facilitates virus membrane fusion and discuss its roles in virus entry.

Bunyavirales ribonucleoproteins: the viral replication and transcription machinery

Abstract The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. The RNA segments of the bunyavirus genome are separately encapsidated by multiple copies of nucleoprotein (N), and both termini of each N-encapsidated genomic RNA segment bind to one copy of the viral L polymerase protein. The viral genomic RNA, N and L protein together form the ribonucleoprotein (RNP) complex that constitutes the molecular machinery for viral genome replication and transcription. Recently, breakthroughs have been achieved in understanding the architecture of bunyavirus RNPs with the determination of the atomic structures of the N and L proteins from various members of this order. In this review, we discuss the structures and functions of these bunyavirus RNP components, as well as viral genome replication and transcription mechanisms.

Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells

Influenza A viruses have sophisticated strategies to promote their own replication. Here, we found that three H5N1 influenza viruses display different replication patterns in human A549 and macrophage cells. The HN01 virus displayed poor replication compared to HN021 and JS01. In addition, the HN01 virus was unable to counteract the interferon response and block general gene expression. This capability was restored by three amino acid substitutions on the NS1 protein: K55E, K66E, and C133F, resulting in recovered binding to CPSF30 and decreased interferon response activity. Furthermore, a recombinant HN01 virus expressing either NS1-C133F or the triple mutation replicate with higher titers in human A549 cells and macrophages compared to the parent virus. These three amino acid mutations reveal a new pathway to alter H5N1 virus replication.

IFN-λ: A new spotlight in innate immunity against influenza virus infection

Influenza virus is a long-lasting and severe threat to human health. Seasonal flu epidemics, which are caused by the cocirculating influenza A viruses (IAVs) and influenza B viruses (IBVs), occur annually and lead to tens of millions of respiratory illnesses and up to half a million human deaths worldwide each year (Ginsberg et al., 2009). Influenza pandemics are more devastating. The 2009 swine-originated H1N1 virus, which caused the latest influenza pandemic, spread from Mexico and U.S. to virtually all countries throughout the world within only several months and was associated much higher mortality among children, young adults, and pregnant women than typical seasonal influenza viruses (Fineberg, 2014). The zoonotic avian influenza viruses, including H5N1, H5N6, H7N9 and H10N8, cause alarmingly high fatality rate in human cases, raising a public concern of pandemic influenza outbreak of avian origin (Poovorawan et al., 2013; Barr, 2017; Bui et al., 2017). Innate immune system is an important barrier of defending against influenza virus infection. According to the traditional paradigm, after IAV gets across the mucus that covers the respiratory epithelium, it first invades and infects respiratory epithelial cells, from where it spreads to other nonimmune and immune cells (e.g., macrophages and dendritic cells). In these cells, the virus can be sensed by the pattern recognition receptors (PRRs), triggering the production of type I interferons (IFNs) which induce the expression of hundreds of IFN-stimulated genes (ISGs) that block viral replication and further virus spread. Simultaneously, activation of PRRs also leads to production of pro-inflammatory cytokines (IL-6, IL-1β, IL-18, TNF, etc.) and chemokines. Pro-inflammatory cytokines induce topical and systemic inflammation, cause fever and anorexia, and direct the adaptive immune response against the virus. Chemokines, on the other hand, recruit innate immune cells (neutrophils, monocytes, and NK cells) which engulf and inactivate the virus, kill virally infected cells, and guide subsequent innate and adaptive immune responses that mediate ultimate viral clearance (Iwasaki and Pillai, 2014). However, effective protection from influenza virus infection is provided by finely tuned antiviral immunity, while excessive innate immunity causes detrimental inflammation. Infection with influenza viruses is usually self-limited, though the severe cases, especially caused by highly virulent strains (e.g., the 2009 pandemic H1N1 virus, H5N1 and H7N9) are characterized by severe pulmonary disease and lethal acute respiratory distress syndrome (ARDS) (Bauer et al., 2006; Ramsey and Kumar, 2011; Ma et al., 2017). Influenza-induced ARDS, which involves the damage to the epithelial-endothelial barrier of the pulmonary alveolus, flute leakage to the alveolar lumen, and respiratory insufficiency, is associated not only with direct viral damage to epithelialendothelial barrier, but also with inflammation mediated by components of the innate immune response. The cytokines produced in influenza virus infected epithelial and endothelial cells, and cytokines and reactive oxygen species produced by neutrophils and macrophages recruited to pulmonary alveolus all contribute to damage to the epithelialendothelial barrier (Short et al., 2014). Both the protective and pathological roles of innate immunity have been evidenced in human and experimental animals. On one hand, genetic deficiency in production of interferon or certain ISGs (e.g., MX and IFITM proteins) increases the vulnerability to IAV infection (Ciancanelli et al., 2015, 2016). On the other hand, genetic ablation of the proinflammatory cytokine IL-6 (Dienz et al., 2012), or chemical depletion of alveolar macrophages (Abboud et al., 2015), NK (Abboud et al., 2015) or neutrophils (Tate et al., 2009) in mice can exacerbate lung injury during IAV infection. These data support that both IFN response and inflammatory innate immune response are essential in protecting the host against IAV infections. However, gene expression analysis displays that early innate immunity signatures including pro-inflammatory cytokines (TNF, IL-1β, IL-6), chemokines (CCL2, CCL3, CCL4, CXCL1) and neutrophil infiltration are strongly associated with acute death of the mice infected with IAV (Brandes et al., 2013). In addition, deletion of pro-