In the past 5 years, an atypical clinical outbreak of avian leukosis virus subgroup J (ALV-J), which contains a unique 205-nucleotide deletion in its 3′ untranslated region (3′UTR), has become epidemic in chickens in China. To determine the role of the 205-nucleotide deletion in the pathogenicity of ALV-J, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated HLJ09SH01) containing the 205-nucleotide deletion in its 3′UTR. The second virus was a chimeric clone in which the 3′UTR contains a 205-nucleotide sequence corresponding to a region of the ALV-J prototype virus. The replication and pathogenicity of the rescued viruses (rHLJ09SH01 and rHLJ09SH01A205) were investigated. Compared to rHLJ09SH01A205, rHLJ09SH01 showed a moderate growth advantage in vitro and in vivo, in addition to exhibiting a higher oncogenicity rate and lethality rate in layers and broilers. Increased vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth receptor subtype 2 (VEGFR-2) expression was induced by rHLJ09SH01 more so than by rHLJ09SH01A205 during early embryonic vascular development, but this increased expression disappeared when the expression levels were normalized to the viral levels. This finding suggests that the expression of VEGF-A and VEGFR-2 is associated with viral replication and may also represent a novel molecular mechanism underlying the oncogenic potential of ALV-J. Overall, our findings not only indicate that the unique 205-nucleotide deletion in the ALV-J genome occurred naturally in China and contributes to increased pathogenicity but also point to the possible mechanism of ALV-J-induced oncogenicity.

Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼10⁴ nanopores per mm² in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.

The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.