Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Outcomes of Paediatric Cystine Stone Management: Results of a Systematic Review.

PURPOSE OF REVIEW: We wanted to analyse the outcomes of surgical (SWL, URS, PCNL) and medical management of cystine stones in the paediatric population in terms of stone-free status and complication rates, based on all the available literature evidence. RECENT FINDINGS: A systematic review of literature was performed for all studies with paediatric cystine stone management. Twelve studies met the eligibility criteria, of which 4 analysed outcomes of SWL, 2 of URS and 3 of PCNL and 3 focused on the effect of either alkalising agents (potassium citrate, citric acid) or cysteine-binding thiol (CBT) agents (tiopronin, penicillamine). The reported SFR in studies ranged from 50 to 83%, 59 to 100% and 63 to 80.6%, with a complication rate of 2.8-51%, 14-27% and 12.9-15.4% with SWL, URS and PCNL, respectively. Paediatric cystine stones treatment should aim at complete stone clearance, preservation of renal function and prevention of further recurrences. SWL achieves inferior results in case of cystine stones. URS and PCNL are safe and effective procedures in the paediatric population, with a low rate of major complications. Adherence to medical prevention therapies may prolong recurrence-free periods.

MicroRNA-18a targeting of the STK4/MST1 tumour suppressor is necessary for transformation in HPV positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy worldwide. They are the aetiological agents of almost all cervical cancers as well as a sub-set of other anogenital and head and neck cancers. Hijacking of host cellular pathways is essential for virus pathogenesis; however, a major challenge remains to identify key host targets and to define their contribution to HPV-driven malignancy. The Hippo pathway regulates epithelial homeostasis by down-regulating the function of the transcription factor YAP. Increased YAP expression has been observed in cervical cancer but the mechanisms driving this increase remain unclear. We found significant down-regulation of the master Hippo regulatory kinase STK4 (also termed MST1) in cervical disease samples and cervical cancer cell lines compared with healthy controls. Re-introduction of STK4 inhibited the proliferation of HPV positive cervical cells and this corresponded with decreased YAP nuclear localization and decreased YAP-dependent gene expression. The HPV E6 and E7 oncoproteins maintained low STK4 expression in cervical cancer cells by upregulating the oncomiR miR-18a, which directly targeted the STK4 mRNA 3'UTR. Interestingly, miR-18a knockdown increased STK4 expression and activated the Hippo pathway, significantly reducing cervical cancer cell proliferation. Our results identify STK4 as a key cervical cancer tumour suppressor, which is targeted via miR-18a in HPV positive tumours. Our study indicates that activation of the Hippo pathway may offer a therapeutically beneficial option for cervical cancer treatment.

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Outcomes and lessons learnt from practice of retrograde intrarenal surgery (RIRS) in a paediatric setting of various age groups: a global study across 8 centres.

PURPOSE: To analyse and report the practice, outcomes and lessons learnt from a global series of retrograde intrarenal surgery (RIRS) in a paediatric multicentre series. METHODS: A retrospective review of anonymized pooled data gathered globally from 8 centres in paediatric patients (≤ 18 years of age) who had renal stones and underwent RIRS from 2015 to 2020 was performed. Patient demographics, perioperative parameters, stone characteristics, complications and stone-free rate (SFR; defined as endoscopically stone free and/or residual fragments < 2 mm on follow up imaging) were analysed. The cohort was stratified into 3 groups by age: < 5 years (Group A), 5-10 years (Group B) and > 10 years (Group C). Overall, post-operative complication rate was 13.7%. Chi-square comparisons were used for categorical variables; analysis of variance (ANOVA) or Kruskal-Wallis tests were used for continuous variables. RESULTS: 314 patients were analysed. The mean age was 9.54 ± 4.76 years. Groups A, B and C had 67 (21.3%), 83 (26.4%) and 164 (52.2%) patients, respectively. Mean stone size was 10.7 ± 4.62 mm. Pre-stenting was performed in 155 (49.4%) of patients, ureteral access sheaths (UAS) was used in 54.5% of patients with majority (71%) utilizing holmium laser for stone fragmentation. All complications were minor (Clavien-Dindo grade 1 and 2). SFR was 75.5%. CONCLUSIONS: RIRS is acceptable as a first-line intervention in the paediatric population with reasonable efficacy and low morbidity. Complications are slightly higher in patients < 5 years of age, which should be taken into account while counselling patients.

Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation.

Hepatitis C virus (HCV) infection remains a major cause of hepatic inflammation and liver disease. HCV triggers NLRP3 inflammasome activation and interleukin-1ß (IL-1ß) production from hepatic macrophages, or Kupffer cells, to drive the hepatic inflammatory response. Here we examined HCV activation of the NLRP3 inflammasome signaling cascade in primary human monocyte derived macrophages and THP-1 cell models of hepatic macrophages to define the HCV-specific agonist and cellular processes of inflammasome activation. We identified the HCV core protein as a virion-specific factor of inflammasome activation. The core protein was both necessary and sufficient for IL-1ß production from macrophages exposed to HCV or soluble core protein alone. NLRP3 inflammasome activation by the HCV core protein required calcium mobilization linked with phospholipase-C activation. Our findings reveal a molecular basis of hepatic inflammasome activation and IL-1ß release triggered by HCV core protein.

Outcomes of ureteroscopy (URS) for stone disease in the paediatric population: results of over 100 URS procedures from a UK tertiary centre.

PURPOSE: To report the outcomes of paediatric ureteroscopy (URS) for stone disease from a specialist endourology centre in the UK. Ureteroscopy for management of stone disease has increased worldwide and is now being done more commonly in the paediatric age group. METHODS: Data were analysed retrospectively from a database maintained between April 2010 and May 2018. Consecutive patients ≤ 16 years of age undergoing semi-rigid or flexible URS for stone disease were included. Stone size and stone-free rate (SFR) were routinely assessed using an ultrasound (USS) and/or plain KUB XR. Complications were graded according to the Clavien-Dindo classification and recorded within 30 days post-procedure and readmissions within 90 days after the procedure were also captured. RESULTS: Over the 8-year period between April 2010 and April 2018, 81 patients with a mean age of 8.8 years (range 18 months-16 years) and a male to female ratio 1:1.1 underwent 102 procedures (1.28 procedure/patient to be stone free). Of the 81 patients, 29 (35.8%) had comorbidities, with 26 (32%) having multiple comorbidities. The mean (± SEM) single and overall stone size was 9.2 mm (± 0.48, range 3-30 mm) and 11.5 mm (± 0.74, range 4-46 mm) respectively, with 22 (27.1%) having multiple stones. Thirty-five (34.7%) had stent in situ pre-operatively. The stone location was in the ureter (26.6%), lower pole (35.4%), and renal pelvis (16.5%), with 22/81(27%) having multiple stones and 21/102 (20.5%) where a ureteral access sheath (UAS) was used. With a mean hospital stay of 1.2 days, the initial and final SFR was 73% and 99%, respectively, and 61/102 (60%) had ureteric stent placed at the end of the procedure. While there were no intra-operative complications, the readmission rate was less than 1% and there were only three early complications recorded. This included a case each of prolonged admission for pain control (grade I), urinary retention (grade II) and post-operative sepsis requiring a brief ITU admission (grade IV). CONCLUSION: Our study demonstrates that in appropriate setting a high stone-free rate can be achieved with minimal morbidity for paediatric patients. There is potentially a need to factor the increasing role of URS in future paediatric urolithiasis guidelines.

Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer.

OBJECTIVE: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. DESIGN AND RESULTS: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. CONCLUSIONS: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion.

UNLABELLED: The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE: The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways.

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

UNLABELLED: Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified. IMPORTANCE: The recent discovery of new relatives of hepatitis C virus (HCV) enables for the first time the study of cross-species determinants shaping hepaciviral pathogenesis. Nonprimate hepacivirus (NPHV) was described to infect horses and represents so far the closest homolog of HCV. Both viruses encode the same viral proteins; however, NPHV protein functions remain poorly understood. In this study, we aimed to dissect NPHV p7 on a structural and functional level. By using various NMR structures of HCV p7 as templates, three-dimensional homology models for NPHV p7 were generated, highlighting conserved residues that are important for ion channel function. A p7-based trans-complementation approach and the construction of NPHV/HCV p7 chimeric viruses showed that the N terminus and transmembrane domains were nonexchangeable. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious viral particles. These results identify species-specific constraints as well as exchangeable determinants in hepaciviral assembly.

Ureteroscopy for Stone Disease in Paediatric Population is Safe and Effective in Medium-Volume and High-Volume Centres: Evidence from a Systematic Review.

PURPOSE OF REVIEW: The incidence of urinary stone disease among the paediatric population is increasing. Whilst there has been a rise in the number of original studies published on ureteroscopy (URS) in children, critical review still remains under-reported. RECENT FINDINGS: A Cochrane style systematic review was performed to identify all original articles on URS (minimum of 25 cases) for stone disease in paediatric patients between Jan. 1996 and Dec. 2016. Based on the number of reported cases, centres were divided into medium (25-49 cases) and high (≥ 50 cases) volume studies. Thirty-four studies (2758 children) satisfied our search criteria and were included in this review. The mean stone size was 8.6 mm with an overall stone-free rate (SFR) of 90.4% (range 58-100). Medium-volume centres reported a mean SFR of 94.1% (range 87.5-100), whilst high-volume centres reported a mean SFR of 88.1% (range 58-98.5). Mean number of sessions to achieve stone-free status in medium-volume and high-volume groups was 1.1 and 1.2 procedures/patient respectively. The overall complication rate was 11.1% (327/2994). Breakdown by Clavien grade was as follows: Clavien I 69% and Clavien II/III 31%. There were no Clavien IV/V complications, and no mortality was recorded across any of the studies. The overall failure to access rate was 2.5% (76/2944). Medium-volume and high-volume studies had overall complication rates of 6.9% (37/530) and 12.1% (287/2222) respectively, but there was no significant difference in major or minor complications between these two groups. Ureteroscopy is a safe and effective treatment for paediatric stone disease. Medium-volume centres can achieve equally high SFRs and safety profiles as high-volume centres. Despite the rarity of paediatric stone disease, our findings might increase the uptake of paediatric URS procedures.

Genotype-specific differences in structural features of hepatitis C virus (HCV) p7 membrane protein.

The 63 amino acid polytopic membrane protein, p7, encoded by hepatitis C virus (HCV) is involved in the modulation of electrochemical gradients across membranes within infected cells. Structural information relating to p7 from multiple genotypes has been generated in silico (e.g. genotype (GT) 1a), as well as obtained from experiments in form of monomeric and hexameric structures (GTs 1b and 5a, respectively). However, sequence diversity and structural differences mean that comparison of their channel gating behaviour has not thus far been simulated. Here, a molecular model of the monomeric GT 1a protein is optimized and assembled into a hexameric bundle for comparison with both the 5a hexamer structure and another hexameric bundle generated using the GT 1b monomer structure. All bundles tend to turn into a compact structure during molecular dynamics (MD) simulations (Gromos96 (ffG45a3)) in hydrated lipid bilayers, as well as when simulated at 'low pH', which may trigger channel opening according to some functional studies. Both GT 1a and 1b channel models are gated via movement of the parallel aligned helices, yet the scenario for the GT 5a protein is more complex, with a short N-terminal helix being involved. However, all bundles display pulsatile dynamics identified by monitoring water dynamics within the pore.

Viroporins: structure, function and potential as antiviral targets.

The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OVs) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OVs lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous OVs are being tested in preclinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain vaccinia virus, has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Relatively few other OVs have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field. This review summarizes the preclinical and clinical experience of OV therapy in the difficult-to-treat area of HCC.

Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

UNLABELLED: Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or "viroporin," whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. CONCLUSION: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA.

Ureteroscopy for stone disease in the paediatric population: a systematic review.

The aim of the present review was to look at the role of ureteroscopy (URS) for treatment of paediatric stone disease. We conducted a systematic review using studies identified by a literature search between January 1990 and May 2013. All English-language articles reporting on a minimum of 50 patients aged ≤ 18 years treated with URS for stone disease were included. Two reviewers independently extracted the data from each study. A total of 14 studies (1718 procedures) were reported in patients with a mean (range) age of 7.8 (0.25-18.0) years. The mean (range) stone burden was 9.8 (1-30) mm and the mean (range) stone-free rate (SFR) 87.5 (58-100)% with initial therapeutic URS. The majority of these stones were in the ureter (n = 1427, 83.4%). There were 180 (10.5%) Clavien I-III complications and 38 cases (2.2%) where there was a failure to complete the initial ureteroscopic procedure and an alternative procedure was performed. To assess the impact of age on failure rate and complications, studies were subcategorized into those that included children with either a mean age <6 years (four studies, 341 procedures) or a mean age >6 years. (10 studies, 1377 procedures). A higher failure rate (4.4 vs 1.7%) and a higher complication rate (24.0 vs 7.1%) were observed in children whose mean age was <6 years. URS for paediatric stone disease is a relatively safe procedure with a reasonably good SFR, but there seems to be a higher failure and complication rate in children aged <6 years.

Ureteroscopy for Paediatric Renal Tract Stones - Outcomes from a Tertiary European Centre.

AIMS: The use of ureteroscopy in treating paediatric stone disease has risen in recent years. We retrospectively reviewed the results of ureteroscopic stone management for our regional paediatric stone service. MATERIAL AND METHODS: Between April 2010 and October 2013, consecutive patients undergoing ureteroscopy and stone fragmentation were identified. Data were recorded from electronic records for patient demographics, pre-operative assessment, stone characteristics, and intra- and post-operative complications. RESULTS: Twenty-one patients (mean age 8.6 years; range: 1.4-16) had 32 procedures in our series (13 males and 8 females). Five (24%) had a metabolic abnormality and 8 (38%) had an anatomical abnormality. The mean initial stone size was 9.6 mm (range: 5-20) and 10 were left sided. Of the 32 procedures, 18 (56%) had a pre-operative stent. A positive pre-operative urine culture was seen in 4 (13%). CT was used in 6 (19%) with the rest having a combination of USS and/or plain KUB. Of these 21 patients, 13 (62%) were stone free after the first procedure, 17 (81%) after a second and 20 (95%) after a third (mean 1.5 procedures/patient). One patient with a 6-mm residual fragment chose to have surveillance. Eighteen (50%) had post-operative stent insertion. The mean length of stay was 1.5 days (range: 0-5). A minor complication (Clavien 1) was observed in 1 patient. No other complications were recorded. CONCLUSIONS: Ureteroscopy for stone disease in children is feasible with a low complication rate and high stone-free rate.

NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system.

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

The role of dopamine D1 and D2 receptors in adolescent methylphenidate conditioned place preference: sex differences and brain-derived neurotrophic factor.

This study analyzed the role of dopamine D1 and D2 receptors in methylphenidate (MPH) conditioned place preference (CPP) in adolescent male and female rats, in addition to the role of these receptors in the effects of MPH on brain-derived neurotrophic factor (BDNF) in the dorsal striatum and nucleus accumbens. Using a nonbiased CPP procedure, the animals were conditioned from postnatal day (PD) 33 to 37. On conditioning trials, animals were first administered saline or their respective antagonist (0.1 or 0.2 mg/kg SCH-23390; 0.01 or 0.03 mg/kg eticlopride HCl), followed by MPH (5 mg/kg). Approximately 10 min after MPH administration, the rats were placed into the paired context for a 10-min trial. One day after conditioning on PD38, a preference test was administered with dividers removed. One day following the preference test on PD39, brain tissue was removed, and the nucleus accumbens and striatum were analyzed for BDNF. Results revealed that MPH conditioning resulted in an increased preference that was blocked by either dose of SCH-23390, but generally not affected by either dose of eticlopride. Further, the higher dose of SCH-23390 resulted in a conditioned place aversion in males, presumably due to an increased number of dopamine D1 receptors in adolescent males. MPH produced a significant increase of striatal and accumbal BDNF alleviated by SCH-23390 or eticlopride. These results show that MPH results in CPP in adolescent male and female rats and these effects appear to be mediated by the dopamine D1 receptor, but the effects of MPH on BDNF appear to be mediated by both dopamine receptor families.