Underwhelming the immune response: effect of slow virus growth on CD8+-T-lymphocyte responses

The speed of virus replication has typically been seen as an advantage for a virus in overcoming the ability of the immune system to control its population growth. Under some circumstances, the converse may also be true: more slowly replicating viruses may evoke weaker cellular immune responses and therefore enhance their likelihood of persistence. Using the model of lymphocytic choriomeningitis virus (LCMV) infection in mice, we provide evidence that slowly replicating strains induce weaker cytotoxic-T-lymphocyte (CTL) responses than a more rapidly replicating strain. Conceptually, we show a "bell-shaped" relationship between the LCMV growth rate and the peak CTL response. Quantitative analysis of human hepatitis C virus infections suggests that a reduction in virus growth rate between patients during the incubation period is associated with a spectrum of disease outcomes, from fulminant hepatitis at the highest rate of viral replication through acute resolving to chronic persistence at the lowest rate. A mathematical model for virus-CTL population dynamics (analogous to predator [CTL]-prey [virus] interactions) is applied in the clinical data-driven analysis of acute hepatitis B virus infection. The speed of viral replication, through its stimulus of host CTL responses, represents an important factor influencing the pathogenesis and duration of virus persistence within the human host. Viruses with lower growth rates may persist in the host because they "sneak through" immune surveillance.     

Cooperative binding promotes demand-driven recruitment of AnxA8 to cholesterol-containing membranes

The functionality of cellular membranes is critically determined by their lipid composition. Within the endolysosomal system, cholesterol is mainly found in more peripheral compartments. In contrast, cholesterol levels are low in late endosomes/lysosomes (LEL), and the occurrence of enlarged pools of this lipid is commonly linked to endolysosomal dysfunction. Here, we show that Annexin A8 (AnxA8), a member of the annexin family of Ca^2 +-dependent membrane-binding proteins, participates in the endosomal regulation of cholesterol homeostasis. Depletion of AnxA8 caused accumulation of cholesterol in LEL, and pharmacological inhibition of the LEL cholesterol export recruited AnxA8 to the cholesterol-laden LEL. Biophysical analysis revealed that cholesterol enhanced the Ca^2 +-dependent affinity of AnxA8 to lipid bilayers, and induced positive cooperativity of membrane binding. Our findings identify AnxA8 as a regulator of LEL cholesterol balance and point to altered membrane binding cooperativity induced by aberrant lipid composition in the target membrane as a means to control the demand-driven recruitment of this cytosolic regulatory protein.     

Inward Rectification in ClC-0 Chloride Channels Caused by Mutations in Several Protein Regions

Several cloned ClC-type Cl⁻ channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl⁻ channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl⁻ concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527–531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463–472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO₄ ⁻) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.     

Overproduction,purification and characterisation of Tbj1, a novel Type III Hsp40 from Trypanosoma brucei,the African sleeping sickness parasite

The heat shock protein 40 (Hsp40) family of proteins act as co-chaperones of the heat shock protein 70 (Hsp70) chaperone family, and together they play a vital role in the maintenance of cellular homeostasis. The Type III class of Hsp40s are diverse in terms of both sequence identity and function and have not been extensively characterised. The Trypanosoma brucei parasite is the causative agent of Human African Trypanosomiasis, and possesses an unusually large Hsp40 complement, consisting mostly of Type III Hsp40s. A novel T. brucei Type III Hsp40, Tbj1, was heterologously expressed, purified, and found to exist as a compact monomer in solution. Using polyclonal antibodies to the full-length recombinant protein, Tbj1 was found by Western analysis to be expressed in the T. brucei bloodstream-form. Tbj1 was found to be able to assist two different Hsp70 proteins in the suppression of protein aggregation in vitro, despite being unable to stimulate their ATPase activity. This indicated that while Tbj1 did not possess independent chaperone activity, it potentially functioned as a novel co-chaperone of Hsp70 in T. brucei.     

Loss of Cytosolic Phosphoglucose Isomerase Affects Carbohydrate Metabolism in Leaves and Is Essential for Fertility of Arabidopsis

Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.Starch and Suc turnover are major pathways of primary metabolism in all higher plants. As such, they are essential for carbohydrate storage and the energy supply of sink tissues and as building blocks for amino acid, fatty acid, or cell wall biosynthesis (Stitt and Zeeman, 2012).A core reaction in both starch and Suc biosynthesis is the reversible interconversion of the hexose phosphate pool metabolites Fru 6-phosphate (Fru6P) and Glc 6-phosphate (Glc6P), which is mediated by phosphoglucose isomerase (PGI). Arabidopsis (Arabidopsis thaliana) contains two isoforms of PGI, one in the plastids and one in the cytosol (Caspar et al., 1985).During the light period, the plastid isoform of PGI (PGI1) is involved in starch biosynthesis by generating Glc6P from the primary photosynthetic product Fru6P. Glc6P is further converted to Glc 1-phosphate (Glc1P) and ADP-glucose via action of phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase), respectively (Stitt and Zeeman, 2012). Finally, transfer of the glucosyl moiety of ADP-glucose to the growing carbohydrate chain of starch is mediated by starch synthases. Any of the enzymatic reactions of this linear pathway is essential for starch synthesis, as illustrated by the virtual absence of transitory starch in chloroplasts of mutant plant lines with impaired function of PGI1 (Yu et al., 2000; Kunz et al., 2010), PGM (Caspar et al., 1985; Kofler et al., 2000), or AGPase (Lin et al., 1988). Interestingly, in a few specific cell types, e.g. leaf guard cells and root columella cells, loss of PGI1 activity can be bypassed by the presence of the plastid Glc6P/phosphate translocator GPT1 (Niewiadomski et al., 2005; Kunz et al., 2010).The cytosolic isoform of PGI (cPGI) is involved in anabolism and catabolism of Suc, the major transport form of carbohydrates in plants. Glc6P and Fru6P interconversion is necessary for both Suc synthesis during the day and during the night. During the day, Suc synthesis in source leaves is fueled mainly by triose phosphates exported from chloroplasts that are eventually converted to Fru6P in the cytosol. However, Fru6P is only one substrate for the Suc-generating enzyme Suc phosphate synthase. The second substrate, UDP-glucose, is synthesized from Fru6P via Glc6P and Glc1P by the cytosolic isoenzymes of PGI1 and PGM as well as UDP-glucose pyrophosphorylase.Because Suc is the major long-distance carbon transport form, its synthesis has to continue throughout the night to supply energy and carbohydrates to all tissues. The nocturnal synthesis of Suc is dependent on breakdown and mobilization of transitory starch from chloroplasts (Zeeman et al., 2007) via export of maltose and Glc (Weber et al., 2000; Niittylä et al., 2004; Weise et al., 2004; Cho et al., 2011). Exported maltose is temporarily integrated into cytosolic heteroglycans (Fettke et al., 2005) mediated by disproportionating enzyme2 (DPE2; Chia et al., 2004; Lu and Sharkey, 2004) yielding Glc and a heteroglycan molecule elongated by an α1-4-bound glucosyl residue. Cytosolic Glc can directly be phosphorylated to Glc6P by the action of hexokinase, while temporarily stored Glc in heteroglycans is released as Glc1P mediated by cytosolic glucan phosphorylase2 (PHS2; Fettke et al., 2004; Lu et al., 2006). Both Glc6P and Glc1P can then be converted to UDP-glucose as during the day.Generation of Fru6P, the second substrate for Suc synthesis, can proceed only to a limited extent from triose phosphates during the night. This limitation is caused mainly by the nocturnal inactivation of Fru 1,6-bisphosphatase (Cséke et al., 1982; Stitt, 1990), a key enzyme in Suc biosynthesis during the day. Hence, in contrast to the situation in the light, cPGI activity is now crucial for providing Fru6P from Glc6P.On the catabolic side, degradation of Suc into its monosaccharides in sink tissues yields both Glc6P and Fru6P, of which only Fru6P can be utilized in glycolytic degradation. Therefore, cPGI is also required for Glc6P conversion to Fru6P in glycolysis, which, in combination with respiration, is the major path of energy production in heterotrophic tissues.Impairment or loss of function of enzymes contributing to the cytosolic hexose phosphate pool has recently been investigated for the Glc1P-forming enzyme PGM (Egli et al., 2010). The Arabidopsis genome encodes three PGM isoforms, with PGM1 localized to plastids and PGM2 and PGM3 localized to the cytosol (Caspar et al., 1985; Egli et al., 2010). Analyses of transfer DNA (T-DNA) mutants showed that homozygous pgm2/pgm3 double mutants were nonviable because of impaired gametophyte development. However, pgm2 and pgm3 single mutants grew like ecotype Columbia (Col-0) wild-type plants, indicating overlapping functions of PGM2 and PGM3 (Egli et al., 2010).By contrast, cPGI is encoded only by a single locus in Arabidopsis (Kawabe et al., 2000). Higher plant mutants reduced in cPGI activity have so far been characterized only in ethyl methanesulfonate-mutagenized Clarkia xantiana (Jones et al., 1986a; Kruckeberg et al., 1989; Neuhaus et al., 1989). The C. xantiana genome encodes for two isoenzymes of cPGI, and homozygous point mutations in each individual cPGI led to significant decrease in cPGI enzyme activity, which was further reduced to a residual activity of 18% in cpgi2/cpgi3 double mutants, where the cPGI3 locus was heterozygous for the mutation (Jones et al., 1986a; Kruckeberg et al., 1989). Detailed physiological analyses of these mutants indicated a negative impact on Suc biosynthesis and elevated starch levels when cPGI activity was decreased at least 3- to 5-fold (Kruckeberg et al., 1989).The physiological impact of decreased or even absent cPGI activity has not been characterized in the genetic model organism Arabidopsis. Here, we show that homozygous T-DNA insertion mutants in the cPGI locus are nonviable and present data from analyses of mature Arabidopsis plants constitutively expressing artificial microRNAs (amiRNAs) targeted against cPGI. These mutants reveal altered photosynthesis, a strong impact on nocturnal leaf starch degradation, and impaired Suc metabolism.     

Uncoupling of Ionic Currents from Substrate Transport in the Plant Ammonium Transporter AtAMT1;2

The ammonium flux across prokaryotic, plant, and animal membranes is regulated by structurally related ammonium transporters (AMT) and/or related Rhesus (Rh) glycoproteins. Several plant AMT homologs, such as AtAMT1;2 from Arabidopsis, elicit ionic, ammonium-dependent currents when expressed in oocytes. By contrast, functional evidence for the transport of NH₃ and the lack of coupled ionic currents has been provided for many Rh proteins. Furthermore, despite high resolution structures the transported substrate in many bacterial homologs, such as AmtB from Escherichia coli, is still unclear. In a heterologous genetic screen in yeast, AtAMT1;2 mutants with reduced transport activity were identified based on the resistance of yeast to the toxic transport analog methylamine. When expressed in oocytes, the reduced transport capacity was confirmed for either of the mutants Q67K, M72I,and W145S. Structural alignments suggest that these mutations were dispersed at subunit contact sites of trimeric AMTs, without direct contact to the pore lumen. Surprisingly, and in contrast to the wild type AtAMT1;2 transporter, ionic currents were not associated with the substrate transport in these mutants. Whether these data suggest that the wild type AtAMT1;2 functions as H⁺/NH₃ co-transporter, as well as how the strict substrate coupling with protons is lost by the mutations, is discussed.     

Role of AMT1;1 in NH4+ acquisition in Arabidopsis thaliana

AtAMT1;1 was the founding member of the family of AMT/Rh ammonium transporters and accounts for about one third of the total ammonium absorption in the roots of the model plant Arabidopsis. Recent evidence suggested that at least some AMT/Rh proteins are NH3 gas channels. In order to evaluate the transported form of ammonium in AtAMT1;1, the protein was functionally expressed in Xenopus oocytes. AtAMT1;1 elicited NH4+ and methylammonium (MeA+) inward currents that saturated in a voltage-dependent manner with a half maximal concentration of 2.7 +/- 1.6 microM for NH4+ and 5.0 +/- 0.7 microM for the transport analogue methylammonium. AtAMT1;1 was plasma membrane localized and expressed in the root cortex and epidermis, including root hairs. The AtAMT1;1-GFP fusion construct under control of its endogenous promoter revealed additional localization of the protein in the pericycle, in the leaf epidermis, and in mesophyll cells. The functional data and its localization suggest that AtAMT1;1 participates in concentrative NH4+ acquisition in roots, in long-distance transport to the shoots, and in re-uptake of apoplastic NH4+ that derives from photorespiration in shoots.