Cell proteins that potentially interact with HBV polymerase were identified by co-immunoprecipitation-based LC-MS/MS identification and IPA / 病毒学报

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.

Identification of ubiquitously expressed transcript as the potential interactor of hepatitis B virus polymerase / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the function of hepatitis B virus polymerase (HBV Pol) in the viral life cycle by screening the proteins interacting with HBV polymerase.</p><p><b>METHODS</b>The HBV Pol gene was constructed into the pGBKT7 vector. GAL4 yeast two-hybrid system was used to screen the human liver cDNA library to obtain proteins which interacted with HBV Pol. GST-pull down assay was applied to confirm the protein interactions.</p><p><b>RESULTS</b>Ubiquitously expressed transcript (UXT) was selected by the yeast two-hybrid system. GST-pull down assay confirmed the in vitro interaction between HBV Pol and UXT.</p><p><b>CONCLUSIONS</b>UXT is a potential interactor of HBV Pol, and this protein interaction may provide clues of the function of HBV Pol in HBV life cycle.</p>

Effect of cell surface sialic acid and their linkages on adhesion of mammary carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of cell surface sialic acid and its linkage on the cell-cell and cell-matrix adhesion of mammary carcinoma cells MD-MB-435.</p><p><b>METHODS</b>MD-MB-435 cells were sense-transfected with ST6Gal I cDNA or antisense-transfected with part of the ST6Gal I sequence inserted in pcDNA 3.1 vector, with mock transfection with pcDNA3.1 vector as the control. The cell surface alpha2, 6-linked sialylation was determined by fluorescence-activated cell sorting (FACS) using lectin SNA (Sambucus nigra agglutinin specific to alpha2, 6-linked sialic acid on N-linked glycoprotein). A significantly increased alpha2, 6-sialylation subclone in sense-transfectants and a decreased alpha2, 6-sialylation subclone in antisense-transfectants were selected for further examination of cell-cell and cell-matrix (collagen IV) adhesion. The transfectants were also treated with sialidase to compare the capacity of cell adhesion affected by cell surface sialylation.</p><p><b>RESULTS</b>Sense-transfection subclone showed a reduced cell-cell aggregation but enhanced cell-matrix adhesion. In contrast, the antisense-transfection subclone exhibited increased cell-cell aggregation and decreased cell-matrix adhesion. After treatment with sialidase, the cell-matrix adhesion of all the transfectants and the parental MDA-MB-435 cells were significantly reduced to the level of 31%-57% of untreated cells.</p><p><b>CONCLUSION</b>Cell surface sialic acid and alpha2, 6-linked sialylation play an important role in cell-cell and cell-matrix adhesion of mammary carcinoma cell MDA-MB-435.</p>