Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.     

Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the K_m values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.     

Comparison of stress responses between mental tasks and white noise exposure

The purpose of this study was to compare the cardiovascular responses to different types of mental stress. Ten healthy males performed a mental arithmetic task (MA) on one day and were exposed to white noise (WN, 80dB) on another day. Both the MA and the WN were composed of four 5-min consecutive periods with a 3-min rest between them. On each day, the systolic and diastolic blood pressure (SBP and DBP), mean arterial pressure (MAP), cardiac output (CO), and total peripheral resistance (TPR) were measured continually during the entire experimental period. The changes from the baseline (Delta) in all periods were calculated for both mental stresses. As for the results, the DeltaMAP, DeltaCO, DeltaHR, and DeltaTPR in the MA did not significantly change during the task periods. However, in the WN, the DeltaMAP and DeltaTPR showed significant increases over the time of the consecutive periods. In addition, we discuss the response patterns for the two mental stresses. We examine three hemodynamic reactivity patterns: a cardiac pattern characterized by increased CO and decreased TPR, a mixed pattern characterized by a moderate increase in both CO and TPR, and a vascular pattern characterized by increased TPR and decreased CO. The results show that throughout all task/exposure periods, the response pattern remained the same for six subjects in each stress. Furthermore, of these six subjects, half showed the same response pattern in both the MA and the WN. In conclusion, compared to the MA task, consecutive WN exposure showed an accumulation of stress responses. A change in TPR contributed to a gradual increase in MAP in the WN. It is also possible that among the subjects there were different types of response to the MA and WN.     

Anti-inflammatory effect of chemically modified chitin

Anti-inflammatory effects of the three types of chitin derivatives namely phosphated chitin (P-chitin), phosphated–sulfated chitin (PS-chitin), and sulfated chitin (S-chitin) were investigated using a canine model of chitosan-induced pneumonia. After simultaneous administration of chitosan with or without each chitin derivative (chitosan alone: n=6, chitosan and P-chitin: n=6, chitosan and PS-chitin: n=1, and chitosan and S-chitin: n=3), hematological examination and X-ray image processing were performed for up to 24 h. Then the lungs were recovered and were evaluated by softex imaging after inflation and fixation. The hematological findings showed that PS-chitin and S-chitin did not prevent the decrease in white blood cell (WBC) count as seen in dogs administered chitosan, while P-chitin prevented such decrease in WBC count. The surface of the inflated and fixed lung specimens was hemorrhagic in the PS- and S-chitin groups as well as in the chitosan group, while the lung looked like normal in the P-chitin group. The pulmonary blood vessels of the chitosan group showed severe change while the P-chitin group showed no changes with softex findings. Furthermore, the pattern of histogram density obtained with image processing of thoracic X-ray in P-chitin group did not change among pre and post administration while chitosan group showed rightward movement and significant changes on parameters. The cause of which is attribured to an attenuation of X-ray permeability by angiectasis of the lung.     

Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.