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1.  Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition 
PLoS Pathogens  2011;7(10):e1002249.
The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.
Author Summary
The influenza neuraminidase (NA) enzyme is the most successful drug target against the seasonal and pandemic flu. The 2009 H1N1 flu pandemic led to record sales of the NA inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza). Recently, a new drug, laninamivir (Inavir), has been approved for use in Japan can also be administered effectively using a single dose via its octanoate prodrug (CS-8958), however its effectiveness against oseltamivir-resistant influenza infection has not been demonstrated in clinical studies. In this study we comprehensively evaluate the effectiveness of laninamivir and its prodrug using NA from different groups with different active site features. We expressed and purified a group 2 NA from the 1957 pandemic H2N2, an atypical group 1 NA from the 2009 H1N1 pandemic and a group 1 NA from avian H12N5. NA inhibition was assayed and NAs were further crystallized with each inhibitor to determine the structural basis of their action. We found that laninamivir inhibition is highly potent for each NA, however binding and inhibition of laninamivir and its prodrug showed group specific preferences. Our results provide the structural and functional basis of NA inhibition using classical and novel inhibitors, with NAs from multiple serotypes with different properties.
PMCID: PMC3197600  PMID: 22028647
2.  Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation 
PLoS Pathogens  2011;7(9):e1002179.
Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.
Author Summary
The fungus Arthrobotrys oligospora has multiple lifestyles. It's not only a nematode pathogen, but also a saprophyte, a pathogen of other fungi, and a colonizer of plant roots. As a nematode pathogen, A. oligospora forms adhesive networks to capture nematodes and is a model organism for understanding the interaction between these fungi and their host nematodes. In this study, the whole genome sequence of A. oligospora was reported. Our analyses of the proteome profiles of intracellular proteins from cells treated with nematode extracts for 10 h and 48 h revealed a key set of genes involved in trap formation. The changes in protein levels for some trap formation related genes were further confirmed by qPCR. The combined genome and proteome analysis identified the major genetic and metabolic pathways involved in trap formation in A. oligospora. Our results provide the first glimpse into the genome and proteome of this fascinating group of carnivorous fungi. The data should serve as a roadmap for further investigations into the interaction between nematode-trapping fungi and their host nematodes, providing broad foundations for research on the biocontrol of pathogenic nematodes.
PMCID: PMC3164635  PMID: 21909256
3.  Cytoplasmic Viral RNA-Dependent RNA Polymerase Disrupts the Intracellular Splicing Machinery by Entering the Nucleus and Interfering with Prp8 
PLoS Pathogens  2014;10(6):e1004199.
The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp) is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3Dpol) also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3Dpol enters the nucleus through the nuclear localization signal (NLS) and targets the pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3Dpol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3Dpol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.
Author Summary
RNA-dependent RNA polymerase (RdRp) is an enzyme that catalyzes the replication from an RNA template and is encoded in the genomes of all RNA viruses. RNA viruses in general replicate in cytoplasm and interfere host cellular gene expression by utilizing proteolytic destruction of cellular targets as the primary mechanism. However, several cytoplasmic RNA viral proteins have been found in the nucleus. What do they do in the nucleus? This study utilized picornaviral polymerase to probe the function of RdRp in the nucleus. Our findings reveal a novel mechanism of viruses attacking hosts whereby picornaviral 3D polymerase (3Dpol) enters the nucleus and targets the central pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The 3Dpol inhibits the second catalytic step of the splicing process, resulting in the accumulation of the lariat-form and the reduction of the mRNA. These results provide new insights into the strategy of a cytoplasmic RNA virus attacking host cell, that differs from viral shutting off cellular transcription and translation which contributes to the viral pathogenesis. To our knowledge, this study shows for the first time that a cytoplasmic RNA virus uses its polymerase to alter cellular gene expression by hijacking the splicing machinery.
PMCID: PMC4072778  PMID: 24968230
4.  Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin 
PLoS Pathogens  2014;10(5):e1004109.
The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.
Author Summary
The staphylococcal phage lysin LysGH15 demonstrates great potential against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the lytic activity of LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, we determined the structures of three individual LysGH15 domains using X-ray crystallography or nuclear magnetic resonance (NMR). The crystal structure unexpectedly reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove in the LysGH15 CHAP domain. Furthermore, the calcium ion plays an important role as a switch that modulates the lytic activity of the CHAP domain. Additionally, structure-guided mutagenesis also confirms that both E282 and the zinc ion play an important role in maintaining the lytic activity of the LysGH15 amidase-2 domain. Moreover, the NMR structure and titration-guided mutagenesis identify residues in the LysGH15 SH3b domain that are involved in the interactions with the substrate. The structure of LysGH15 is the first determined lysin structure from a staphylococcal phage, and these results represent a pivotal step forward in understanding this type of lysin.
PMCID: PMC4022735  PMID: 24831957
5.  Transovarial Transmission of a Plant Virus Is Mediated by Vitellogenin of Its Insect Vector 
PLoS Pathogens  2014;10(3):e1003949.
Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV–planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.
Author Summary
Numerous parasites including viruses, bacteria, and microsporidia can be maternally transmitted, with the parasite passing from mother to offspring, usually through eggs. However, the process of the parasites spreading into eggs from primarily infected tissues and the factors that mediate this process in live hosts or vectors are unknown due to the lack of useful tools. Here, we used several techniques to investigate the molecular mechanisms of transovarial transmission of Rice stripe virus (RSV), a plant virus belonging to the genus Tenuivirus, by its insect vector (Laodelphax striatellus). We found that the nucleocapsid protein of RSV bound to insect's vitellogenin (Vg) in vitro and in vivo. We also found that RSV invaded the egg tubes of the ovariole until Vg is highly expressed, then colocalized with Vg in the germarium. When Vg expression was knocked down due to RNA interference, the invasion of ovarioles by RSV decreased largely. Our study provides new insights into the transovarial transmission of an important viral pathogen that uses existing transovarial transportation systems in insect vectors to invade eggs.
PMCID: PMC3946389  PMID: 24603905
6.  Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention 
PLoS Pathogens  2014;10(2):e1003931.
C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.
Author Summary
Dengue virus (DENV), a mosquito-borne flavivirus, is currently the most significant arbovirus afflicting tropical and sub-tropical countries worldwide. No vaccine or therapeutics are available, and dengue has rapidly spread over the last decade. Therefore, additional strategies to combat dengue are urgently needed. In this study, we characterized multiple C-type lectins as susceptibility factors for dengue infection in A. aegypti. These mosGCTLs directly interacted with dengue virus in vitro and in vivo. The combination of antisera against multiple mosGCTLs efficiently reduced DENV-2 infection after a blood meal, suggesting that it is feasible to develop a mosGCTL-based transmission-blocking vaccine to interrupt the life cycle of dengue virus and control disease burden in nature. This study substantially extends our understanding of dengue replication in vectors and provides a research avenue by which the development of therapeutics for preventing the dissemination of mosquito-borne viral diseases can be pursued in the future.
PMCID: PMC3923773  PMID: 24550728
7.  Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity 
PLoS Pathogens  2013;9(10):e1003659.
Tm-22 is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-22 and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-22-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-22. Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-22 and is required for Tm-22-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.
Author Summary
Plant pathogens cause considerable yield losses in many vegetables and crops; therefore, understanding the mechanisms of disease resistance can enable crop improvements and provide substantial economic benefits. Here, we examine the signaling pathways of Tm-22, a tomato resistance protein that confers resistance to Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by detecting the presence of their movement proteins (MPs). We found that in Nicotiana benthamiana, Tm-22-mediated resistance against TMV and ToMV requires MIP1s (NbMIP1s), a group of J-domain proteins that may act as molecular chaperones to assist protein folding and maintain protein structure. Intriguingly, MIP1s are required for both TMV resistance and TMV infection. NbMIP1s interact with and are essential for protein stability of both Tm-22 and the viral MP. NbMIP1s are required for resistance mediated by other resistance proteins and resistance to the bacterial nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. These new insights into the mechanisms of viral infection and disease resistance may enable future advances in crop protection.
PMCID: PMC3789785  PMID: 24098120
8.  The Smallest Capsid Protein Mediates Binding of the Essential Tegument Protein pp150 to Stabilize DNA-Containing Capsids in Human Cytomegalovirus 
PLoS Pathogens  2013;9(8):e1003525.
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV contains a much larger dsDNA genome within a similarly-sized capsid compared to the others, and it was proposed to require pp150, a tegument protein only found in cytomegaloviruses, to stabilize its genome-containing capsid. However, little is known about how pp150 interacts with the underlying capsid. Moreover, the smallest capsid protein (SCP), while dispensable in herpes simplex virus type 1, was shown to play essential, yet undefined, role in HCMV infection. Here, by cryo electron microscopy (cryoEM), we determine three-dimensional structures of HCMV capsid (no pp150) and virion (with pp150) at sub-nanometer resolution. Comparison of these two structures reveals that each pp150 tegument density is composed of two helix bundles connected by a long central helix. Correlation between the resolved helices and sequence-based secondary structure prediction maps the tegument density to the N-terminal half of pp150. The structures also show that SCP mediates interactions between the capsid and pp150 at the upper helix bundle of pp150. Consistent with this structural observation, ribozyme inhibition of SCP expression in HCMV-infected cells impairs the formation of DNA-containing viral particles and reduces viral yield by 10,000 fold. By cryoEM reconstruction of the resulting “SCP-deficient” viral particles, we further demonstrate that SCP is required for pp150 functionally binding to the capsid. Together, our structural and biochemical results point to a mechanism whereby SCP recruits pp150 to stabilize genome-containing capsid for the production of infectious HCMV virion.
Author Summary
Human cytomegalovirus (HCMV) causes birth defects in newborns and life-threatening complications in immunocompromised individuals, such as AIDS patients and organ transplant recipients. The smallest capsid protein (SCP) – only 8 kDa molecular mass as compared to the 155 kDa major capsid protein – has been demonstrated to be essential for HCMV growth, but is dispensable in herpes simplex virus type 1. These seemingly contradictory observations have been a paradox. Here, we solve this paradox by high resolution cryo electron microscopy (cryoEM), in conjunction with functional studies using ribozyme inhibition. Our structural comparisons of HCMV virion and capsid reveal molecular interactions at the secondary structure level and suggest that SCP might contribute to capsid binding of pp150, an essential, cytomegalovirus-specific tegument protein. SCP-deficient particles generated by ribozyme inhibition of SCP-expression in HCMV-infected cells show no pp150 tegument density, demonstrating that SCP is required for the functional binding of pp150 to the capsid. Our results suggest that SCP recruits pp150 to stabilize the HCMV nucleocapsid to enable encapsidation of the genome, which is more densely packaged in HCMV than in other herpesviruses. Overall, this study not only resolves the above paradox, but also illustrates the passive acquisition of a new, essential function by SCP in the production of infectious HCMV virions.
PMCID: PMC3744435  PMID: 23966856
9.  HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1 
PLoS Pathogens  2013;9(4):e1003248.
Upon recognition of viral components by pattern recognition receptors, such as the toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases, cells are activated to produce type I interferon (IFN) and proinflammatory cytokines. These pathways are tightly regulated by the host to prevent an inappropriate cellular response, but viruses can modulate these pathways to proliferate and spread. In this study, we revealed a novel mechanism in which hepatitis C virus (HCV) evades the immune surveillance system to proliferate by activating microRNA-21 (miR-21). We demonstrated that HCV infection upregulates miR-21, which in turn suppresses HCV-triggered type I IFN production, thus promoting HCV replication. Furthermore, we demonstrated that miR-21 targets two important factors in the TLR signaling pathway, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are involved in HCV-induced type I IFN production. HCV-mediated activation of miR-21 expression requires viral proteins and several signaling components. Moreover, we identified a transcription factor, activating protein-1 (AP-1), which is partly responsible for miR-21 induction in response to HCV infection through PKCε/JNK/c-Jun and PKCα/ERK/c-Fos cascades. Taken together, our results indicate that miR-21 is upregulated during HCV infection and negatively regulates IFN-α signaling through MyD88 and IRAK1 and may be a potential therapeutic target for antiviral intervention.
Author Summary
Hepatitis C virus (HCV), a major cause of chronic hepatitis, end-stage cirrhosis, and hepatocellular carcinoma, has chronically infected 200 million people worldwide and 3–4 million more each year. When triggered by viral infection, host cells produce type I interferon (IFN) and proinflammatory cytokines to antagonize the virus. Despite extensive research, the mechanism underlying HCV immune system evasion remains elusive. Our results provided the first direct evidence that microRNA-21 (miR-21) feedback inhibits type I IFN signaling when cells are challenged with HCV, thus promoting the infection. MicroRNA is a kind of endogenous non-coding small RNA that regulates a wide range of biological processes and participate in innate and adaptive immune responses through complementarily pairing with target mRNA, which can regulate its expression or translation. Currently, miRNAs have intrigued many scientists as potent targets or therapeutic agents for diseases. In our study, the targets of miR-21, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are important for HCV-induced type I IFN production, have also been found. Moreover, we identified a transcription factor, AP-1, which is partly responsible for miR-21 induction in response to HCV infection. Taken together, our research has provided new insights into understanding the effects of miRNA on host-virus interactions, and revealed a potential therapeutic target for antiviral intervention.
PMCID: PMC3635988  PMID: 23633945
10.  Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography 
PLoS Pathogens  2013;9(1):e1003132.
Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.
Author Summary
Rabbit hemorrhagic disease (RHD), first described in China in 1984, causes hemorrhagic necrosis of the liver within three days after infection and with a mortality rate that exceeds 90%. RHD has spread to large parts of the world and threatens the rabbit industry and related ecology. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. Currently, the absence of a high-resolution model of any lagovirus impedes our understanding of its molecular interactions with hosts and successful design of an efficient anti-RHDV vaccine. Here, we use hybrid structural approaches to construct a pseudo-atomic model of RHDV that reveals significant differences in the P2 sub-domain of the major capsid protein compared to that seen in other caliciviruses. We identified seven regions of high sequence variation in this sub-domain that dictate the binding specificities of histo-blood group antigens. In one of these regions, we identified an antigenic peptide that interacts with rabbit tissue cells and elicits a significant immune response in rabbits and, hence, protects them from RHDV infection. Our pseudo-atomic model provides a structural framework for developing new anti-RHDV vaccines and will also help guide use of the RHDV capsid as a vehicle to display human tumor antigens as part of anti-tumor therapy.
PMCID: PMC3547835  PMID: 23341770
11.  The ESCRT Machinery Is Recruited by the Viral BFRF1 Protein to the Nucleus-Associated Membrane for the Maturation of Epstein-Barr Virus 
PLoS Pathogens  2012;8(9):e1002904.
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
Author Summary
Herpesviruses are large DNA viruses associated with human and animal diseases. After viral DNA replication, the herpesviral nucleocapsids egress through the nuclear membrane for subsequent cytoplasmic virion maturation. However, the mechanism by which the virus regulates the nuclear membrane and cellular machinery involved in this process remained elusive. The cellular endosomal sorting complex required for transport (ESCRT) machinery is known to participate in the biogenesis of multivesicular bodies, cytokinesis and the release of enveloped viruses from cytoplasmic membranes. Here, we show that functional ESCRT machinery is required for the maturation of Epstein-Barr virus (EBV). ESCRT proteins are redistributed close to the nucleus-associated membrane through interaction with the viral BFRF1 protein, leading to vesicle formation and structural changes of the nuclear membrane. Remarkably, inhibition of ESCRT machinery abolishes BFRF1-induced vesicle formation, and leads to the accumulation of viral DNA and capsid proteins in the nucleus. Specific interactions between BFRF1 and Alix are required for BFRF1-derived vesicle formation and crucial for the nuclear egress of EBV.
PMCID: PMC3435242  PMID: 22969426
12.  Crystal Structures Reveal the Multi-Ligand Binding Mechanism of Staphylococcus aureus ClfB 
PLoS Pathogens  2012;8(6):e1002751.
Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.
Author Summary
Staphylococcus aureus (S. aureus), an important opportunistic pathogen, is a major threat to humans and animals, causing high morbidity and mortality worldwide. It is responsible for a variety of infections ranging from mild superficial infections to severe infections such as infective endocarditis, septic arthritis, osteomyelitis and sepsis. Such infections are of growing concern due to the increasing antibiotic resistance of S. aureus. In order to understand the mechanism of the S. aureus pathogenesis, we studied one of the bacterial surface proteins clumping factor B (ClfB) bound by the fibrinogen α (Fg α) and cytokeratin 10 (CK10). From analyses of the high resolution crystal structures we found that the ClfB-binding peptides harbor a stretch with consensus sequence (GSSGXGXXG) that is also conserved in Engrailed protein, TCF20 and Dermokines. The interaction between ClfB and a dermokine-derived peptide was demonstrated using binding assays. Consistent with a role of ClfB in the inflammatory responses induced by S. aureus, expression of dermokines is predominant in epithelial tissues and upregulated in inflammatory diseases. The data presented in this study raise a possibility that multiple human proteins are targeted by ClfB during S. aureus infection. The multi-ligand binding feature of ClfB would be valuable for developing new therapeutic strategies.
PMCID: PMC3375286  PMID: 22719251
13.  Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance 
PLoS Pathogens  2012;8(6):e1002752.
Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.
Author Summary
Plants utilize a multilayered immune system to protect themselves against pathogens. One layer of innate immunity is controlled by intracellular immune receptors called disease resistance (R) proteins. Plant R proteins are powerful molecules capable of triggering host cell suicide thereby restricting pathogen growth. Therefore, it is crucial for plants to control R protein activity in signaling cell death to avoid harmful autoimmune responses. The Barley MLA locus encodes a number of immune receptors that each recognizes a specific powdery mildew fungal strain. Upon pathogen recognition MLAs trigger host defenses concomitant with a rapid cell death response. We here show that MLA10 cell death-inducing activity is tightly regulated by conserved motifs located in two of its domains and by specific cellular chaperone components. Furthermore, we show distinct functions for the nuclear and cytoplasmic MLA10 pools in disease resistance and cell death signaling and provide evidence for a model uncoupling MLA10 cell death signaling from its disease resistance activity. Our results suggest that plant immune receptors integrate signals from multiple sub-cellular compartments to coordinate effective immune responses against pathogen attack.
PMCID: PMC3369952  PMID: 22685408
14.  Epstein-Barr Virus-Encoded LMP1 Interacts with FGD4 to Activate Cdc42 and Thereby Promote Migration of Nasopharyngeal Carcinoma Cells 
PLoS Pathogens  2012;8(5):e1002690.
Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility— cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis.
Author Summary
Epstein-Barr virus (EBV) is closely associated with human malignancies, including nasopharyngeal carcinoma (NPC). Among EBV-expressed genes, latent membrane protein 1 (LMP1) has been detected in most NPC tissues and has the ability to transform cell growth and drive cell migration, both of which are highly associated with tumorigenesis and tumor progression. Previous reports have demonstrated that cell migration primarily involves cytoskeleton rearrangement, and the RhoGTPase Cdc42 is known to actively mediate such rearrangement processes. Using LMP1-expressing NPC cells, we discovered that LMP1 induces Cdc42 activation by directly binding to FGD4, a positive regulator of Cdc42, thereby promoting motility of NPC cells. The observed correlation between FGD4 and LMP1 expression in NPC tissues provides support of physiological relevance. Notably, FGD4 has recently been shown to be responsible for a type of inherited neural disease. Our findings not only provide a novel insight into EBV pathogenesis, but also suggest a role for FGD4 in tumorigenesis.
PMCID: PMC3349753  PMID: 22589722
15.  Mitochondrial Ubiquitin Ligase MARCH5 Promotes TLR7 Signaling by Attenuating TANK Action 
PLoS Pathogens  2011;7(5):e1002057.
The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-κB-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63-linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling.
Author Summary
In 2005, MAVS was characterized as the critical adaptor protein for the signal transduction of RIG-I-like receptors (RLRs). This provided the first link between mitochondria and the intracellular antiviral defense system. From then on, exploring the potential functions of novel mitochondrial proteins in microbe-host interactions became a rapidly expanding frontier. Notably, it remains unknown whether mitochondrial proteins can directly regulate TLR signaling. Here, we demonstrate that the mitochondrial protein MARCH5 positively modulates TLR7 signaling. Our study reveals that MARCH5 is a novel E3 ubiquitin ligase and catalyzes the K63-linked poly-ubiquitination of TANK. This modification releases the inhibitory effects of TANK on TRAF6. Arguably, this represents the first study linking mitochondria to TLR signaling, shedding new light on the role of mitochondria in the proinflammatory response.
PMCID: PMC3098239  PMID: 21625535
16.  Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity 
PLoS Pathogens  2010;6(7):e1001018.
Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world's population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4+ and CD8+ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV+ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8− Jurkat cells and CD4+ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.
Author Summary
Hepatitis C Virus (HCV) is a major public health concern with a large number of individuals infected (3% world wide). Currently, there is no effective vaccine available to prevent HCV infection and the treatment is effective in less than half of all patients. Therefore, many patients have long term infections that lead to severe liver damage or liver cancer. It has been shown that some HCV infected patients can eliminate the virus and the host immune system is involved. The problem is most people do not have the capacity to fight their HCV infection. We have developed a gene therapy based approach where a patient's own immune cells can be made to recognize cells expressing HCV genes. This can be accomplished regardless of his or her natural capacity to fight their HCV infection. This manuscript describes how normal immune cells can be genetically altered to recognize cells expressing HCV proteins and characterizes their reactivity and sensitivity to antigen stimulation.
PMCID: PMC2912399  PMID: 20686664
17.  Protective Efficacy of Centralized and Polyvalent Envelope Immunogens in an Attenuated Equine Lentivirus Vaccine 
PLoS Pathogens  2015;11(1):e1004610.
Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.
Author Summary
Our best effort for containment of the global HIV epidemic is the development of a broadly protective vaccine. Current research has focused on vaccines that can generate a protective immune response against numerous strains of the virus. For this reason, vaccines with centralized HIV genes as immunogens, which merge HIV genetic information and potentially protect against multiple viral strains in a single inoculation, are an increasing area of interest to the field. Existing published studies have not evaluated centralized immunogens in the context of attenuated vaccines, which to date, have demonstrated the highest level of vaccine protection in lentiviral studies. Furthermore, centralized immunogen studies have also not included protective efficacy findings accomplished through challenge with highly pathogenic virus strains. In this study we not only examine the immunogenicity of these immunogens in an animal model, but we also for the first time evaluate the ability of centralized immunogens to induce protection against virulent virus challenge.
PMCID: PMC4287611  PMID: 25569288
18.  Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses 
PLoS Pathogens  2014;10(11):e1004508.
H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.
Author Summary
Avian influenza viruses continue to present challenges to human health. Recently the H7N9 and H10N8 viruses that are of low pathogenicity for poultry have caused human infections and deaths in China. H9N2 influenza virus have been isolated worldwide from wild and domestic avian species for several decades, and their low pathogenic nature to poultry made them a low priority for animal disease control, which has allowed them to continue to evolve and spread. Here, we investigated a series of H9N2 influenza viruses that were detected in live poultry markets in southern China. We found that these viruses are able to preferentially bind to the human-type receptor, and some of them can cause disease and transmit between ferrets by respiratory droplet. All the transmissible H9N2 viruses have a similar internal gene constellation, which was also present in the H7N9 and H10N8 viruses. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.
PMCID: PMC4239090  PMID: 25411973
19.  Densovirus Is a Mutualistic Symbiont of a Global Crop Pest (Helicoverpa armigera) and Protects against a Baculovirus and Bt Biopesticide 
PLoS Pathogens  2014;10(10):e1004490.
Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus.
Author Summary
The old world cotton bollworm, Helicoverpa armigera, is one of the most significant pests of crops throughout Asia, Europe, Africa and Australia. Herein, we report a novel densovirus (HaDNV-1) which was widely distributed in wild populations of H. armigera and was beneficial to its host by increasing larval and pupal development rates, female lifespan and fecundity, suggesting a mutualistic interaction between the cotton bollworm and HaDNV-1. The cotton bollworm is currently widely controlled by the biopesticides Bacillus thuringiensis (Bt) toxin and the baculovirus HaNPV. It is therefore important to estimate the risk that the symbiotic virus will negatively impact on the efficiency of these biopesticides. Field and laboratory results suggest that HaDNV-1 infection significantly increases larval resistance to HaNPV and Bt toxin. These results have important implications for the selection of biopesticides for this species, and highlight the need for greater research into the elegant microbial interactions that may impact host individual and population dynamics.
PMCID: PMC4214819  PMID: 25357125
20.  The pH-Responsive PacC Transcription Factor of Aspergillus fumigatus Governs Epithelial Entry and Tissue Invasion during Pulmonary Aspergillosis 
PLoS Pathogens  2014;10(10):e1004413.
Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.
Author Summary
Inhaled spores of the pathogenic mould Aspergillus fumigatus cause fungal lung infections in humans having immune defects. A. fumigatus spores germinate within the immunocompromised lung, producing invasively growing, elongated cells called hyphae. Hyphae degrade the surrounding pulmonary tissue, a process thought to be caused by secreted fungal enzymes; however, A. fumigatus mutants lacking one or more protease activities retain fully invasive phenotypes in mouse models of disease. Here we report the first discovery of a non-invasive A. fumigatus mutant, which lacks a pH-responsive transcription factor PacC. Using global transcriptional profiling of wild type and mutant isolates, and in vitro pulmonary invasion assays, we established that loss of PacC leads to a compound non-invasive phenotype characterised by deficits in both contact-mediated epithelial entry and protease expression. Consistent with an important role for epithelial entry in promoting invasive disease in mammalian tissues, PacC mutants remain surface-localised on mammalian epithelia, both in vitro and in vivo. Our study sets a new precedent for involvement of both host and pathogen activities in promoting epithelial invasion by A. fumigatus and supports a model wherein fungal protease activity acting subsequently to, or in parallel with, host-mediated epithelial entry provides the mechanistic basis for tissue invasion.
PMCID: PMC4199764  PMID: 25329394
21.  Cyclophilin A Associates with Enterovirus-71 Virus Capsid and Plays an Essential Role in Viral Infection as an Uncoating Regulator 
PLoS Pathogens  2014;10(10):e1004422.
Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.
Author Summary
Enterovirus 71 (EV71) is the major causative agent of hand-foot-and-mouth disease (HFMD) in Asia-Pacific region and caused over one million infection cases and nine hundred deaths in the year of 2010 in China mainland. EV71 is known to infect the young children for the sake of their undeveloped immune system. Unlike other Enterovirus (e.g. coxsackievirus), EV71 could cause severe aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis, thus leading to high fatality rates. There is no clinically applied therapeutics. In this work, we used CypA inhibitors as bioprobes to show that CypA played an essential role in EV71 proliferation. We also elucidated the mechanism by which CypA interacted with the EV71 VP1 H-I loop and functioned as an uncoating regulator in EV71 entry step. Since there are several non-immunosuppressive CypA inhibitors, e.g. NIM-811 and Debio-025, have been reported to show antiviral potency, our results provide a potential way to discover clinical therapeutics against EV71 infection.
PMCID: PMC4183573  PMID: 25275585
22.  Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus 
PLoS Pathogens  2014;10(10):e1004420.
Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.
Author Summary
The identification of influenza virus-like sequences in two different bat species has generated great interest in understanding their biology, ability to mix with other influenza viruses, and their public health threat. Unfortunately, bat-influenza viruses couldn't be cultured from the samples containing the influenza-like nucleic acids. We used synthetic genomics strategies to create wild type bat-influenza, or bat-influenza modified by substituting the surface glycoproteins with those of model influenza A viruses. Although influenza virus-like particles were produced from both synthetic genomes, only the modified bat-influenza viruses could be cultured. The modified bat-influenza viruses replicated efficiently in vitro and an H1N1 modified version caused severe disease in mice. Collectively our data show: (1) the two bat-flu genomes identified in other studies are replication competent, suggesting that host cell specificity is the major limitation for propagation of bat-influenza, (2) bat-influenza NS1 antagonizes host interferon response more efficiently than that of a model influenza A virus, (3) bat-influenza has both genetic and protein incompatibility with influenza A or B viruses, and (4) that these bat-influenza lineages pose little pandemic threat.
PMCID: PMC4183581  PMID: 25275541
23.  Reactive Oxygen Species Regulate Caspase-11 Expression and Activation of the Non-canonical NLRP3 Inflammasome during Enteric Pathogen Infection 
PLoS Pathogens  2014;10(9):e1004410.
Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3- and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway.
Author Summary
Caspase-11 is required for NLRP3 inflammasome activation and cell death in response to certain gram-negative bacterial infections like Citrobacter rodentium. However, how C. rodentium drives caspase-11 expression and activation is not well understood. Here, we demonstrate that the NOD2-RIP2 pathway regulates reactive oxygen species production and c-Jun N-terminal kinase signaling to control caspase-11 expression and subsequent activation of caspase-11 and the NLRP3 inflammasome during C. rodentium infection. In the absence of NOD2-RIP2 signaling, increased inflammasome activation results in lower bacteria numbers in the colon and less tissue damage during the early stages of infection.
PMCID: PMC4178001  PMID: 25254654
24.  MHC Class II Restricted Innate-Like Double Negative T Cells Contribute to Optimal Primary and Secondary Immunity to Leishmania major 
PLoS Pathogens  2014;10(9):e1004396.
Although it is generally believed that CD4+ T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3+CD4−CD8− (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.
Author Summary
Although it is generally believed that CD4+ T cells mediate anti-Leishmania immunity, some studies suggest that CD3+CD4−CD8− (double negative, DN) T cells may play a more important role in regulating primary anti-Leishmania immunity. Here, we report that DN T cells extensively proliferate and produce effector cytokines in mice following primary and secondary L. major infections. Leishmania-reactive DN T cells utilize αβ T cell receptor (TCR) and are restricted by MHC class II molecules. Strikingly, DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation, effector cytokine production in vitro and in vivo, and accelerated parasite control following secondary L. major challenge. These results directly identify DN T cells as important players in protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.
PMCID: PMC4169504  PMID: 25233487
25.  Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli 
PLoS Pathogens  2014;10(9):e1004404.
Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.
Author Summary
Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhea worldwide and is commonly present as an infection in symptomatic travelers returning from developing countries. The attachment of EAEC to the human intestine is mediated protein filaments extending from the bacterial surface known as aggregative adherence fimbria (AAF). Here we use X-ray crystallography and nuclear magnetic resonance (NMR) structures to provide an atomic structure of the protein fibers made by the two major variants, AAF/I and AAF/II. The structures of the major subunit proteins show that the AAFs assemble into flexible, linear polymers that are capped by a single minor protein subunit at the tip. Biochemical assays reveal that the AAFs recognize a common receptor, the extracellular matrix protein fibronectin, via clusters of positively-charged amino acid residues running along the length of the fimbriae. Our structures suggest a unique mechanism based on ionic interactions for AAF-mediated receptor binding and biofilm formation.
PMCID: PMC4169507  PMID: 25232738

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