Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships

Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal ‘stem’ of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system

Tumor expression of survivin, p53, cyclin D1, osteopontin and fibronectin in predicting the response to neo-adjuvant chemotherapy in children with advanced malignant peripheral nerve sheath tumor

Purpose Selected cell-cycle regulators and extracellular matrix proteins were found to play roles in malignant peripheral nerve sheath tumor (MPNST) biology. We aimed to analyze whether initial tumor tissue expressions of survivin, p53, cyclin D1, osteopontin (OPN) and fibronectin (FN) correlate with the response to neo-adjuvant CHT (naCHT) in children with advanced inoperable MPNST. Methods The study included 26 children with MPNST (M/F 14/12, median age 130 months) treated in Polish centers of pediatric oncology between 1992 and 2013. Tissue expression of markers was studied immunohistochemically in the manually performed tissue microarrays and assessed semi-quantitatively as low and high, based on the rate of positive cells and staining intensity. Results Good response to naCHT was noted in 47.6%, while poor-in 52.4% of patients. The response to naCHT was influenced negatively by the presence of neurofibromatosis NF1 and high initial tumor tissue expression of OPN, survivin, p53 and cyclin D1. Patients with high tumor expression of either OPN, survivin or p53 and those with simultaneous high expression of ≥ 3 of the markers, responded significantly worse to naCHT, than patients, in whom expression of ≤ 2 markers were detected at diagnosis. Nearly, 85% of patients expressing ≥ 3 markers, responded poor to CHT; while 87.5% of children, expressing ≤ 2 markers, were good responders. Conclusion The initial tumor tissue expression of OPN, survivin, p53 and cyclin D1 may serve as markers to predict response to naCHT in pediatric advanced MPNST. Future studies in more numerous group of patients are needed to confirm these preliminary results

Hepatitis C - new developments in the studies of the viral life cycle

Hepatitis C virus (HCV) is a causative agent of chronic liver disease leading to cirrhosis, liver failure and hepatocellular carcinoma. The prevalence of HCV is estimated as 3% of the world population and the virus is a major public health problem all over the world. For over 16 years, since HCV had been discovered, studies of the mechanisms of the viral life cycle and virus-host interactions have been hampered by the lack of a cell culture system allowing the virus to be grown in laboratory conditions. However, in recent years some new model systems to study HCV have been developed. The major breakthrough of the last two years was the cell culture system for maintaining the virus in an adapted hepatocyte-derived cell line. This review describes the techniques and applications of most of the in vitro systems and animal models currently used for working with hepatitis C virus

Antivirals - current trends in fighting influenza

Influenza virus infection is a major source of morbidity and mortality worldwide. Due to the variable effectiveness of existing vaccines, especially in the early stages of an epidemic, antiviral drugs represent the first line of defense against the virus. Currently, there are two major classes of anti-influenza drugs approved by the FDA for clinical use: M2 protein inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (zanamivir and oseltamivir). However, increasing resistance to these available influenza antivirals among circulating influenza viruses highlights the need to develop alternative approaches for the prevention and/or treatment of influenza. This review presents an overview of currently available drugs for influenza treatment as well as summarizes some new antiviral strategies that are now being tested covering agents targeting both the viral proteins and the host-virus interaction. We discuss their mechanisms of action, resistance and the therapeutic potential as new antiviral drug for use in future influenza pandemics. Additionally, combination therapy based on these drugs is also described

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies▿ †

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1WT, the JFH1N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1T416A and JFH1I422L more efficiently than the WT virus, while neutralization of JFH1N417S and JFH1G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1N415D, highlighting the importance of the E2 412-423 region in virus entry