Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.     

水稻草矮病毒NS6基因在大肠杆菌中的表达及植物表达载体的构建

Large amount of disease-specific protein(SP) accumulated in the rice plant cells infected by rice grassy stunt virus(RGSV). It was deduced that the protein was encoded by NS6 gene on genomic vRNA6 and thus referred to as NS6 protein.But its function is unknown. In an effort to prove the above deduction and to elucidate the function of NS6 protein of RGSV, we constructed a bacterial expression plasmid pGTNS6 producing a fusion protein of glutathione S-transferase (GST) and NS6 protein, and a plant expression vector pCBTNSv6 containing NS6 gene. A recombinant plasmid pTNSv 6 containing the coding region of NS6 gene and the non-coding region at its 5' terminus, cloned by RT-PCR from purified RNAs of Shaxian isolate of RGSV, was used as the start point. Western blot analysis showed that the fusion protein reacted strongly with antisera raised against RGSV-SP, which served as evidence of the deduction.EHA105 of Agrobacterium tumefasciens containing pCBTNSv6 has been obtained and the transformation of rice is underway.     

Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.     

Virus adaptation to quantitative plant resistance: erosion or breakdown?

Adaptation of populations to new environments is frequently costly due to trade‐offs between life history traits, and consequently, parasites are expected to be locally adapted to sympatric hosts. Also, during adaptation to the host, an increase in parasite fitness could have direct consequences on its aggressiveness (i.e. the quantity of damages caused to the host by the virus). These two phenomena have been observed in the context of pathogen adaptation to host's qualitative and monogenic resistances. However, the ability of pathogens to adapt to quantitative polygenic plant resistances and the consequences of these potential adaptations on other pathogen life history traits remain to be evaluated. Potato virus Y and two pepper genotypes (one susceptible and one with quantitative resistance) were used, and experimental evolutions showed that adaptation to a quantitative resistance was possible and resulted in resistance breakdown. This adaptation was associated to a fitness cost on the susceptible cultivar, but had no consequence either in terms of aggressiveness, which could be explained by a high tolerance level, or in terms of aphid transmission efficiency. We concluded that quantitative resistances are not necessarily durable but management strategies mixing susceptible and resistant cultivars in space and/or in time should be useful to preserve their efficiency.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.     

Are chlorella viruses a rich source of ion channel genes?

Plaque-forming dsDNA (>330 kb) viruses that infect certain unicellular, eukaryotic chlorella-like green algae contain approximately 375 protein-encoding genes. These proteins include a 94 amino acid K+ channel protein, called Kcv, as well as two putative ligand-gated ion channels. The viruses also encode other proteins that could be involved in the assembly and/or function of ion channels, including protein kinases and a phosphatase, polyamine biosynthetic enzymes and histamine decarboxylase.