Purification of hepatitis C virus core protein in non-denaturing condition.

Hepatitis C virus (HCV) is the major cause of chronic hepatitis and hepatocellular carcinoma. Among its structural proteins, the HCV core protein has been implicated in liver disease. Understanding the role of HCV core proteins in viral diseases is crucial to elucidating disease mechanisms and identifying potential drug targets. However, purification challenges hinder the comprehensive elucidation of the structure and biochemical properties of HCV core proteins. In this study, we successfully solubilized bacterially expressed core protein using a high-salt and detergent-containing buffer and bypassed the denaturing-refolding process. Size-exclusion chromatography revealed three distinct peaks in the HCV-infected cell lysate, with the bacterially expressed soluble core protein corresponding to its second peak. Using a combination of affinity, size exclusion, and multi-modal chromatography purification techniques, we achieved a purity of > 95% for the core protein. Analytical ultracentrifugation revealed monomer formation in the solution. Far UV Circular dichroism spectroscopy identified 25.53% alpha helices and 20.26% beta sheets. These findings strongly suggest that the purified core proteins retained one of the native structures observed in HCV-infected cells.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

X-ray crystallographic structure of RNase Po1 that exhibits anti-tumor activity.

RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus ostreatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disulfide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic potential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein.

Serum levels of tenascin-C in collagen diseases.

Tenascins are a family of large multimetric extracellular matrix (ECM) proteins. Among them, large molecular weight variant tenascin-C is known to be specifically expressed in pathological conditions. However, no link between tenascin-C and collagen diseases has been established. The aim of our study was to determine the serum tenascin-C levels in patients with various collagen diseases, and to evaluate the possibility that serum levels of tenascin-C can be a useful marker for collagen diseases, correlating with the pathogenesis. Serum tenascin-C levels of 33 patients with scleroderma (SSc), 10 patients with scleroderma spectrum disorder (SSD), 15 patients with localized scleroderma (LSc), 12 patients with dermatomyositis (DM), 10 patients with systemic lupus erythematosus (SLE) and 15 healthy controls were measured with specific enzyme-linked immunosorbent assays. Serum tenascin-C levels were significantly elevated in patients with SSc, SSD and LSc than in healthy controls. Significantly higher total skin thickness score or higher incidence of pitting scars/ulcers and diffuse pigmentation were observed in SSc patients with elevated tenascin-C levels than in those with normal levels. Our study suggests that serum tenascin-C levels are increased in fibrotic conditions, and that tenascin-C contributes to the pathogenesis of vascular damage as well as fibrosis in SSc patients. Clarifying the role of tenascin-C in the pathogenesis of collagen diseases may lead to a new therapeutic approach.

Retroviral integration site analysis and the fate of transduced clones in an MDR1 gene therapy protocol targeting metastatic breast cancer.

A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.