Challenges and opportunities for early-career Teaching-Focussed academics in the biosciences.

Twenty-seven percent of academics in UK Higher Education (HE) are in Teaching-Focussed positions, making major contributions to undergraduate programmes in an era of high student expectations when it comes to teaching quality. However, institutional support for Teaching-Focussed academics is often limited, both in terms of peer networking and opportunities for career development. As four early-career stage Teaching-Focussed academics working in a variety of institutions, we explore what motivated our choices to make teaching our primary academic activity, and the challenges that we have faced in doing so. In addition to highlighting the need for universities to fully recognise the achievements of teaching staff, we discuss the role that the various biosciences learned societies have in supporting Teaching-Focussed academics. We identify that there is a need for the learned societies to come together and pool their expertise in this area. The fragmented nature of the Teaching-Focussed academic community means that clear sources of national support are needed in order to best enable the next generation of bioscience educators to reach their full potential.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase.

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Hepatitis C virus RNA replication is regulated by Ras-Erk signalling.

The hepatitis C virus NS5A protein has been previously demonstrated to partially attenuate activation of the Ras-Erk signalling pathway, via a conserved class II polyproline motif located towards the C terminus of the protein. However, the role of Ras-Erk signalling in the virus life cycle remains undetermined. To investigate this, levels of RNA replication were measured in genotypes 1 and 2 transient luciferase subgenomic replicon systems in the context of either pharmacological or genetic (dominant-negative) inhibition of MEK1, a kinase in the Ras-Erk signalling cascade. Incubation in the presence of two inhibitors (U0126 and PD184352) resulted in a decrease in the levels of RNA replication, conversely incubation with inhibitor PD98059 resulted in a modest increase in replication. The results obtained with PD98059 could not be explained by an off-target effect on Cox-2, stability of replicon RNA or stimulation of global translation levels, suggesting stimulation by a yet uncharacterized mechanism. To verify data obtained using pharmacological inhibitors the transient replicon RNA was co-electroporated with a dominant-negative mutant of MEK1. This resulted in a reduction in replication, confirming data seen with U0126 and PD184352. Our data are consistent with the hypothesis that a low level Ras-Erk signalling activity is required for RNA replication. However, complete inhibition of Ras-Erk signalling is inhibitory. These results suggest that perturbation of this signalling pathway by NS5A may be a mechanism to regulate levels of genomic RNA replication.

A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly.

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

Mobility analysis of an NS5A-GFP fusion protein in cells actively replicating hepatitis C virus subgenomic RNA.

We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A-GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A-GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.

Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci.

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP-NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.