Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background

Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.

Principal Findings

After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.

Conclusions

Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.     

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.     

Pull-push neuromodulation of LTP and LTD enables bidirectional experience-induced synaptic scaling in visual cortex

Neuromodulatory input, acting on G protein-coupled receptors, is essential for the induction of experience-dependent cortical plasticity. Here we report that G-coupled receptors in layer II/III of visual cortex control the polarity of synaptic plasticity through a pull-push regulation of LTP and LTD. In slices, receptors coupled to Gs promote LTP while suppressing LTD; conversely, receptors coupled to Gq11 promote LTD and suppress LTP. In vivo, the selective stimulation of Gs- or Gq11-coupled receptors brings the cortex into LTP-only or LTD-only states, which allows the potentiation or depression of targeted synapses with visual stimulation. The pull-push regulation of LTP/LTD occurs via direct control of the synaptic plasticity machinery and it is independent of changes in NMDAR activation or neuronal excitability. We propose these simple rules governing the pull-push control of LTP/LTD form a general metaplasticity mechanism that may contribute to neuromodulation of plasticity in other cortical circuits.     

Molecular Mechanisms Endowing Cross-resistance to ALS-Inhibiting Herbicides in Amaranthus hybridus from Argentina

Amaranthus hybridus L. is one of the most problematic weeds in summer crops in Argentina. However, 20 years after the detection of the first case of resistance to ALS-inhibiting herbicides in this country, no extensive reports of the molecular mechanisms endowing resistance were published. In this work, we sequenced the acetolactate synthase gene of resistant plants belonging to five different populations of A. hybridus from Santa Fe and Cordoba provinces. We found that every population presented at least one of the previously documented substitutions W574L and D376E in ALS amino acid sequence. These results explain the cross-resistance to ALS-inhibiting herbicides and should alert about the usage of herbicides with a different site of action after an ineffective control of this species. This is the first report of these target-site mechanisms endowing resistance to ALS-inhibiting herbicides in A. hybridus populations from Argentina.

     

Kv7 channels can function without constitutive calmodulin tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> extracellular adherence protein domain 4

The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. Here we report the backbone and side-chain resonance assignments of the 11.3 kDa fourth domain of Eap. The assignment data has been deposited in the BMRB database under the accession number 26726.     

In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures

We describe herein the antitrypanosomal activity of 20 novel 1,3-bis(aryloxy)propan-2-amine derivatives. Compounds 2, 4, 6, 12, 15, 16 and 19 were significantly active against amastigote and trypomastigote forms, with half maximal inhibitory concentrationvalues in the range of 6-18 µM. In the cytotoxicity tests against L929 cells, only compound 4 presented selectivity index value above 10, indicating low toxicity.