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.

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles.

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving trans-complementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focus-forming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence.

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria.

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine.

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.

The 'hook effect' on serum prolactin estimation in a patient with macroprolactinoma.

Large quantities of antigen in an immunoassay system impair antigen-antibody binding, resulting in low antigen determination. This is called the 'high dose hook effect'. We report this phenomenon in a patient with a large macroprolactinoma. In this patient, the correct estimate of serum prolactin (PRL) was obtained only after appropriate dilution of serum. We suggest that in order to avoid the high dose hook effect, the serum PRL be estimated in appropriate dilution in all patients with large pituitary tumours. This is particularly important when the clinical suspicion of high PRL is strong, as in women with amenorrhoea-galactorrhoea and men with long standing hypogonadism.

Predictive value of serum thyroglobulin in treated patients with differentiated thyroid carcinoma.

An in-house radioimmuno assay for serum thyroglobulin was developed in our laboratory and compared its relative sensitivity with that of whole body scan in the detection of residual tumour or metastases and evaluated the predictive value of serum thyroglobulin in the clinical course of the disease. Ninety six patients after thyroidectomy were followed up for a maximum period of five years in this study.The sensitivity and specificity of serum thyroglobulin were found to be close to that of whole body scan (85% and 94% respectively). According to this study, a serum thyroglobulin >40 ng/ml can differentiate between patients with metastases and those in remission. Serum thyroglobulin can replace whole body scan during the subsequent follow-up if the patient had concordant whole body scan and serum thyroglobulin during initial assessment.

Thyroid autoantibody measurement by enzyme immunoassay.

Thyroid antibodies are commonly utilized in the assessment and diagnosis of autoimmune thyroid disorders. We compared the measurements of antithyroglobulin and antithyroidperoxidase antibodies by enzyme immunoassay with that of the conventional agglutination method. This fully automated enzyme immunoassay is more specific and cost effective than the agglutination method. Further this is a very quantitative and rapid method producing results in two hours as compared to at least twenty=four hours required by the conventional method. Antithyroidperoxidase antibodies determined by enzyme immunoassay are more specific and sensitive in the diagnosis of Hashimoto's thyroiditis than the antithyroglobulin antibodies.

Thyroid stimulating hormone receptor antibody in thyroid diseases.

Thyroid stimulating hormone receptor antibody (TRA) was estimated as a measure of TSH binding inhibitory immunoglobulin (TBII) in 48 persons. These included (i) 14 controls; (ii) 23 patients with Graves' disease who were tested for TRA within 3 months of commencing treatment with carbimazole of which 13 were studied serially; (iii) 5 patients with toxic nodular goitre; (iv) 4 with euthyroid exophthalmos; and (v) 2 neonates of thyrotoxic mothers. TRA was measured with an RIA system, while total thyroxine (T4), free thyroxine concentration (FTC) and TSH were also estimated along with TRA. All controls showed undetectable TRA levels; 87 per cent of patients with Graves' disease were TRA positive within 3 months of starting carbimazole therapy. In the serial study, 5 patients with Graves' disease who had undetectable TRA initially remained so while on treatment. Seven out of the remaining 8 patients showed a decline of TRA levels to normal over 3 to 18 months. This decline coincided with clinical and biochemical recovery.