Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus

ABSTRACT Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion

The Role Of The Membrane Proximal Region Of The M2 Cytoplasmic Tail In Virus Replication

Influenza A virus encodes M2, a proton channel that has been shown to be important during virus entry and assembly. The primary aim of this thesis was to investigate the role of the membrane proximal region, residues 46-69, of the M2 cytoplasmic tail during virus replication. A cholesterol recognition/interaction amino consensus: CRAC) motif, previously identified in the membrane proximal region of M2 in some influenza A virus strains, was suggested to play a role in virus replication by mediating incorporation of M2 into budding virus particles. Alteration or completion of the M2 CRAC motif in two different recombinant virus strains caused no changes in virus replication in tissue culture; however, viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection. In order to further investigate the role of the membrane proximal residues of M2 in basic virus replication, scanning and directed alanine mutants were generated and analyzed in trans-complementation assays and recombinant viruses. The membrane proximal residues 46-69 tolerated numerous mutations with little, if any, affect on virus replication suggesting that the identity of individual amino acids in this region are less important than the overall protein structure for the M2 protein function. The requirement during virus replication of the ectodomain and the cytoplasmic tail of M2, which includes the membrane proximal region, was further characterized using the influenza C virus CM2 protein and a chimeric influenza A virus M2 protein: MCM) containing the CM2 transmembrane domain. While M2, CM2, and MCM could all alter cytosolic pH to varying degrees when expressed from cDNA, only M2 and MCM could at least partially complement an M2-null virus in a trans-complementation system. This data suggests that while the CM2 ion channel activity is similar to that of M2, sequences in the ectodomain and/or cytoplasmic tail play important roles in infectious virus production. This thesis suggests that the structure of the membrane proximal region of the M2 cytoplasmic tail may stabilize the membrane distal region, which mediates genome incorporation

Host Range, Biology, and Species Specificity of Seven-Segmented Influenza Viruses—A Comparative Review on Influenza C and D

Other than genome structure, influenza C (ICV), and D (IDV) viruses with seven-segmented genomes are biologically different from the eight-segmented influenza A (IAV), and B (IBV) viruses concerning the presence of hemagglutinin–esterase fusion protein, which combines the function of hemagglutinin and neuraminidase responsible for receptor-binding, fusion, and receptor-destroying enzymatic activities, respectively. Whereas ICV with humans as primary hosts emerged nearly 74 years ago, IDV, a distant relative of ICV, was isolated in 2011, with bovines as the primary host. Despite its initial emergence in swine, IDV has turned out to be a transboundary bovine pathogen and a broader host range, similar to influenza A viruses (IAV). The receptor specificities of ICV and IDV determine the host range and the species specificity. The recent findings of the presence of the IDV genome in the human respiratory sample, and high traffic human environments indicate its public health significance. Conversely, the presence of ICV in pigs and cattle also raises the possibility of gene segment interactions/virus reassortment between ICV and IDV where these viruses co-exist. This review is a holistic approach to discuss the ecology of seven-segmented influenza viruses by focusing on what is known so far on the host range, seroepidemiology, biology, receptor, phylodynamics, species specificity, and cross-species transmission of the ICV and IDV

Characterisation of the human signal peptidase complex as a quality control enzyme for membrane proteins

During membrane protein biogenesis, cells need to detect and degrade faulty proteins. Despite a key role in cellular homeostasis and human diseases, little is known about the underlying mechanisms. In recent years, few endoplasmic reticulum (ER)-resident proteases have been linked to quality control by cleaving their clients and thereby facilitating membrane extraction and degradation via the ER-associated degradation (ERAD) pathway. The major ER-resident protease in mammalian cells is the signal peptidase complex (SPC), a tetra subunit complex discovered in the 1970s to be responsible for the removal of signal sequences from ER-targeted and secretory proteins. Until now, this was thought to be the only function of the SPC besides a few studies reporting a role in the maturation of viral polyproteins. In this work, I show that the SPC also acts as a membrane protein quality control factor. First, through proteome-wide computational analyses, I identified approximately 1500 membrane proteins containing SPC cryptic cleavage sites after N-terminal and internal type-II oriented transmembrane domains (TMDs). I then validated SPC cleavage for several candidate substrates (Cx32, Cx26, Cx30.3, PMP22, iRhom2 and Hrd1) and revealed that SPC cleavage relies on the accessory subunit SPCS1 as recognition factor to discern between signal sequences and TMDs. Moreover, I show that the SPC cleaves membrane proteins when they fail to fold properly or assemble correctly into their native complexes, thus exposing cryptic cleavage sites. I also show that this SPC cleavage mechanism cooperates with the ERAD pathway to help maintain a functional membrane proteome and confers a fitness advantage to cells exposed to ER stress. Finally, I report first data on the possible role of the SPC in controlling protein abundance beyond its quality control function. Overall, this thesis characterises a novel function of the SPC, expanding its substrate spectrum, extending the knowledge on the essential cellular functions performed by this protease and laying the foundations for future work at the organismal level in quality control-related diseases and beyond

The Biology of the Influenza D Virus

Influenza D virus (IDV) was first identified in 2011 from clinically ill pigs in America. In nearly a decade since its discovery, the virus has been detected in multiple animal species in a vast region of the globe and is considered an important cause of concern to animal and human health. IDV utilizes cattle as the primary reservoir. The viral infection can cause mild respiratory disease in cattle and has been indicated as a causative agent of bovine respiratory disease (BRD) complex that is the most common and costly disease affecting the cattle industry. Moreover, outbreaks of IDV in swine and bovine are increasing, and more genetic and serological evidences show that IDV has a potential to adapt to humans. IDV is unique among four types of influenza viruses. The thermal and acid stability of IDV were examined and directly compared with those of influenza A virus (IAV), influenza B virus (IBV), and influenza C virus (ICV). The results of our experiments demonstrated that only IDV had a high residual infectivity (~2.5 log units of 50% tissue culture infective dose ([TCID50]/ml) after a 60-min exposure to 53°C in solution at a neutral pH, and remarkably, IDV retained this infectivity even after exposure to 53°C for 120 min. Furthermore, the data showed that IDV was extremely resistant to inactivation by low pH. After being treated at pH 3.0 for 30 min, IDV lost only approximately 20% of its original infectiousness, while all other types of influenza viruses were completely inactivated. Finally, replacement of the hemagglutinin (HA) and neuraminidase (NA) proteins of a temperature- and acid-sensitive IAV with the hemagglutinin-esterase fusion (HEF) protein of a stable IDV through a reverse genetic system largely rendered the recombinant IAVs resistant to high-temperature and low-pH treatments. Together, these results indicated that the HEF glycoprotein is a primary determinant of the exceptional temperature and acid tolerance of IDV. Further investigation into the viral entry and fusion mechanism mediated by the intrinsically stable HEF protein of IDV may offer novel insights into how the fusion machinery of influenza viruses evolve to achieve acid and thermal stability, which as a result promotes the potential to transmit across mammal species. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmid based system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replication-incompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3’ conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient crossrecognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reverse-genetics system that can be used to further study replication, tropism, and pathogenesis of IDV. Based on the sequences of the hemagglutinin-esterase-fusion (HEF) gene, IDV can be classified into three genetic lineages: D/OK-lineage, D/660-lineage and D/Japan-lineage. The D/swine/Oklahoma/1334/2011 (D/OK) and D/Bovine/Oklahoma/660/2013 (D/660) are the representative strains of the D/OK-, and D/660 -lineages, respectively. We found that the replication of the D/OK virus was approximately 2 log10TCID50/ml lower than that of the D660 virus in different cell lines. Interestingly, by using our reverse genetics system, we generated recombinant chimeric D/OK viruses in that one of each genomic segments was replaced with the segment from the D660 virus, and observed that only the replication fitness of the chimeric D/OK virus with the D660 NP segment was significantly increased. Finally, we identified two positions 247 and 381 within the NP protein were key determinants of the replication difference between the D/OK and D660 viruses. Interestingly, theses amino acid changes in the NP had no effect on IDV RNP activity but may affect virus replication in the late stage of the viral life cycles, which warrants further investigation

Genetic profiling of Mycoplasma hyopneumoniae

A microarray was constructed and applied to transcriptional profiling and genetic variation studies of Mycoplasma hyopneumoniae. The genome sequence enabled the construction of the microarray to allow a global approach to understanding fundamental processes in M. hyopneumoniae . These studies focused on whether M. hyopneumoniae regulates its genes under different environmental conditions and if genetic changes can be correlated with virulence. The microarray consisted of 632 open reading frames represented by polymerase chain reaction products and were used in a two-color experimental design. Data were analyzed using a mixed linear statistical model. Unique features implemented in these studies included the printing of two complete arrays per substrate, reducing the slide to slide variation; the scanning of each dye channel of each array at different laser power settings to increase the dynamic range of expression measurement; and the use of a unique set of hexamer primers to generate fluorescently labeled targets for microarray analysis. The first series of studies focused on transcriptional profiling during heat shock and iron deprivation, two environmental changes that M. hyopneumoniae encounters on the respiratory epithelial surface during disease. Key genes responsive to these stresses were identified, and interestingly, fifty-three were regulated in common to the two conditions at p\u3c0.05. Since adherence to swine cilia is a prerequisite for colonization and disease, the next series of studies involved the comparison of an adherent, pathogenic strain 232 to a nonpathogenic, nonadherent type strain J. In conjunction with this study, two strain 232 adherence variants, one high and one low, were also compared. Thirty genes were identified that differed between the strains 232 and J. Nineteen genes were up-regulated in the high adherent variant including the heat shock protein DnaJ, and fifteen genes were up-regulated in the low adherent strain. In a comparison of eight field isolates to strain 232, two were indistinguishable from strain 232 and the others varied in an many as twenty-five loci and as few as one. In summary, these studies attempt to unravel some of the underlying mechanisms and pathways M. hyopneumoniae uses to infect the host and cause disease

New Advances on Zika Virus Research

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that historically has been associated with mild febrile illness. However, the recent outbreaks in Brazil in 2015 and its rapid spread throughout South and Central America and the Caribbean, together with its association with severe neurological disorders—including fetal microcephaly and Guillain-Barré syndrome in adults—have changed the historic perspective of ZIKV. Currently, ZIKV is considered an important public health concern that has the potential to affect millions of people worldwide. The significance of ZIKV in human health and the lack of approved vaccines and/or antiviral drugs to combat ZIKV infection have triggered a global effort to develop effective countermeasures to prevent and/or treat ZIKV infection. In this Special Issue of Viruses, we have assembled a collection of 32 research and review articles that cover the more recent advances on ZIKV molecular biology, replication and transmission, virus–host interactions, pathogenesis, epidemiology, vaccine development, antivirals, and viral diagnosis

Plant Viruses: From Ecology to Control

Plant viruses cause many of the most important diseases threatening crops worldwide. Over the last quarter of a century, an increasing number of plant viruses have emerged in various parts of the world, especially in the tropics and subtropics. As is generally observed for plant viruses, most of the emerging viruses are transmitted horizontally by biological vectors, mainly insects. Reverse genetics using infectious clones—available for many plant viruses—has been used for identification of viral determinants involved in virus–host and virus–vector interactions. Although many studies have identified a number of factors involved in disease development and transmission, the precise mechanisms are unknown for most of the virus–plant–vector combinations. In most cases, the diverse outcomes resulting from virus–virus interactions are poorly understood. Although significant advances have been made towards understand the mechanisms involved in plant resistance to viruses, we are far from being able to apply this knowledge to protect cultivated plants from the all viral threats.The aim of this Special Issue was to provide a platform for researchers interested in plant virology to share their recent results. To achieve this, we invited the plant virology community to submit research articles, short communications and reviews related to the various aspects of plant virology: ecology, virus–plant host interactions, virus–vector interactions, virus–virus interactions, and control strategies. This issue contains some of the best current research in plant virology