Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system

“Natural” Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na+ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na+ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na+ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Chronic stress alters the immune response to influenza virus vaccine in older adults.

To determine whether a chronic stressor (caregiving for a spouse with a progressive dementia) is associated with an impaired immune response to influenza virus vaccination, we compared 32 caregivers' vaccine responses with those of 32 sex-, age-, and socioeconomically matched control subjects. Caregivers showed a poorer antibody response following vaccination relative to control subjects as assessed by two independent methods, ELISA and hemagglutination inhibition. Caregivers also had lower levels of in vitro virus-specific-induced interleukin 2 levels and interleukin 1beta; interleukin 6 did not differ between groups. These data demonstrate that down-regulation of the immune response to influenza virus vaccination is associated with a chronic stressor in the elderly. These results could have implications for vulnerability to infection among older adults.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation.

The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable. HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide. They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Thesis (Ph.D.)--University of Washington, 2016-06

Morbi-mortalidade por doenças do aparelho respiratório em idosos antes e após a introdução da vacina contra influenza: município de Cubatão, São Paulo, 1999-2005

Apresenta-se análise da morbi-mortalidade por Doenças do Aparelho Respiratório entre idosos de Cubatão e cobertura vacinal no período 1999-2005. Observa-se tendência de redução, acompanhada de aumento da cobertura de vacinação contra o vírus influenza

Newcastle disease virus in penguins from King George Island on the Antarctic region

Here we report the isolation of Newcastle disease virus (NDV) from cloacal swabs obtained from penguins in the South Atlantic Antarctic region (62 degrees 08S, 58 degrees 25W). Samples of 100 penguins from King George Island were tested by real-time PCR, of which 2 (2%) were positive for NDV. The positive samples were isolated in embryonated chicken eggs and their matrix and fusion proteins genes were partially sequenced. This was complemented by the serological study performed on the blood of the same specimens, which resulted in a 33.3% rate of positivity. (C) 2010 Elsevier B.V. All rights reserved.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.