Research progress in membrane fusion process among Paramyxoviruses / 中华实验和临床病毒学杂志

The family Paramyxoviridae is a group of viruses that significantly affect human and animal health. Paramyxoviruses are generally thought to enter host cells by direct fusion of the viral and host cell membranes. Membrane fusion, essential for syncytium formation, is not only a pathological hallmark of paramyxoviral infections, but also a kind of mechanism of cell-to-cell viral spread. Nevertheless, the process of membrane fusion is highly conserved, only consisting of two-component fusion apparatus: an attachment protein (HN/G/H) and a fusion (F) protein. However, there is a significant knowledge gap on the mechanism(s) by which HN/H/G couples receptor binding to F-triggering, since different attachment proteins possess special triggering process. In this review, we summarize the general property and distinction of the fusion process during paramyxoviral infection for further research on the pathogenic mechanism.

Research progress in interaction mechanism of enveloped glycoproteins of human parainfluenza virus type 3 / 中华实验和临床病毒学杂志

Human parainfluenza viruses (hPIVs), a series of single-stranded RNA viruses of Paramyxoviridae, are the main pathogen of respiratory tract infection. hPIV3 is the main cause of lower respiratory tract infection leading to bronchiolitis and pneumonias in young children under the age of six months, and it is the second major pathogen only next to respiratory syncytial virus (RSV). In this paper, we mainly discuss two kinds of virulence-related surface glycoprotein of hPIV3: hemagglutinin-neuraminidase (HN) protein and fusion protein (F) by briefly introducing the protein structure and physiological functions of HN and F. According to the latest research progress, we focus on the models which have been postulated to explain how F and HN work in concert to bring about membrane fusion.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Silencing of Fanconi Anemia Complementation Group F Exhibits Potent Chemosensitization of Mitomycin C Activity in Breast Cancer Cells / 한국유방암학회지

PURPOSE: Fanconi anemia complementation group F (FANCF) is a key factor to maintaining the function of Fanconi anaemia/BRCA (FA/BRCA) pathway, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In the present study, we evaluated the chemosensitization effect of FANCF in breast cancer cells. METHODS: We performed specific knockdown of the endogenous FANCF in breast cancer cells by transfecting the cells with an FANCF short hairpin RNA (shRNA) vector. Cell viability was measured with a Cell Counting Kit-8, and DNA damage was assessed with the alkaline comet assay. The apoptosis, cell cycle, and drug accumulation were measured by flow cytometric analysis. Protein expression levels were determined by Western blot analysis, using specific antibodies. RESULTS: The analyses of two breast cancer cell lines (MCF-7 and MDA-MB-435S) demonstrated that the FANCF shRNA could effectively block the FA/BRCA pathway through the inhibition of Fanconi anemia complementation group D2 ubiquitination. Moreover, FANCF silencing potentiated the sensitivity of cells to mitomycin C (MMC), where combined FANCF shRNA/MMC treatment inhibited cell proliferation, induced S-phase arrest, apoptosis, and DNA fragmentation, and reduced the mitochondrial membrane potential, compared with MMC treatment alone. CONCLUSION: Taken together, this study demonstrates that the inhibition of FANCF by its shRNA leads to a synergistic enhancement of MMC cytotoxicity in breast cancer cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of breast cancer.

Fanconi anemia protein and acute myeloid leukemia / 白血病·淋巴瘤

Fanconi anemia (FA) is an rare autosomal recessive inherited disease which manifests progressive marrow failure, congenital bone malformation, high risk to cancers and so on. Chromatin of FA cells display auto-instability and high hypersensitivity to interstrand DNA cross-links such as mitomycin C. As normally FA develop into acute myeloid leukemia easily, it has been regarded as pre-leukemia state. Till now 11 FA genes have been found and play a role in sustaining stability of gene groups through the same mechanism. As an active connecting protein, FANCF protein play an important part in correct FA complex formation. Which makes FANCD2 single ubiquitin. Ubiquitin FANCD2 induces chromatin and BRCA1 interact, and repair injured DNA. FA gene defect makes gene group instable and increases the risk of chromatin collapse, which finally leads to acute myeloid leukemia and myelodysplastic syndrome.

Influence of F protein of hepatitis C virus subtype 1b inhibits on human hepatocellular carcinom HepG2 cell apoptosis / 中华流行病学杂志

Objective To investigate the effects of F protein of hepatitis C virus subtype lb on the apoptosis of human hepatocellular carcinom HepG2 cells. Methods HepG2 cells were transfected with recombinant plasmid pcDNA3.0-F-EGFP and pcDNA3.0-F-EGFP-HepG2 strain was exposed to Act-D and tumor necrosis factor a (TNFα) treatment in order to induce cell apoptosis with positive control pcDNA3.0-C-EGFP-HepG2, negative control pcDNA3.0-C-EGFP-HepG2 and blank control HepG2.Annexin V-FITC/PI of flow cytometry was performed to determine the number of apoptotic cells. DNA Ladder was used to observe the isolation of apoptotic DNA fragments in the apoptotie cells. Results pcDNA3.0-F-EGFP- HepG2 cell strain showed a much delayed apoptosis as well as obviously lowering the apoptotic rate when compared with the pcDNA3.0-HepG2 strain and HepG2 strain (P<0.001).Conclusion The introduction and expression of extraneous gene (the F gene of hepatitis C virus subtype 1b) could significantly inhibit the apoptosis of HepG2 cells.

Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV)

Human Respiratory Syncytial Virus (HRSV) was first characterized in 1957 and has since been recognized as the most common viral cause of severe respiratory tract infection in young infants worldwide. Despite many years of research there is still no effective treatment or any immediate prospect of a vaccine. The HRSV genome is composed of single stranded negative sense RNA and the virion consists of a nucleocapsid packaged within a lipid envelope. The envelope contains spike-like projections, each being a homo-oligomer of one of three transmembrane viral envelope proteins: the attachment protein G, the fusion protein F involved in viral penetration and the small hydrofobic protein SH. The aim of this work was to construct two recombinant replication-defective adenoviruses carrying separately F and G genes from HRSV. This system was chosen because adenovirus delivers genes into target cells with high efficiency in a variety of cell lines and can be used in vitro and in vivo. In order to obtain the recombinant viruses, we did RT-PCR of RNA extracted from the HRSV A2 strain, the genes F and G were cloned in to pAdeno-X vectors. pAdeno-F and pAdeno-G were transfected in HEK-293 cells for the production of recombinant viruses, that expressed efficiently these two proteins and provide us the means for doing functional assays and immunization tests.

O Vírus Sincicial Respiratório Humano (HRSV) foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment"), a proteína de fusão F ("fusion") e a proteína SH ("small hydrofobic"). O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.

Screening and cloning of the target genes transactivated by HCV FTP2 protein using suppression subtractive hybridization technique / 中华传染病杂志

Objective To clone and identify human genes transactivated by homo sapiens HCV FTP2 by constructing a cDNA subtractive library with suppression subtractive hybridization tech- nique.Methods Suppression subtractive hybridization(SSH)and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV FTP2.The mRNA was iso- lated from HepG2 cells transfected pcDNA3.1(-)-HCV FTP2 and pcDNA3.1(-)empty vector re- spectively,and SSH method was employed to analyze the differentially expressed DNA sequence be- tween the two groups.After digestion with restriction enzyme Rsa I,small-size cDNAs were ob- tained.Then tester cDNA was divided into two groups and ligated to the specific adaptor I and adap- tor 2 respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5?.Futhermore,the cDNA was se- quenced and analyzed in GenBank with Blast search after PCR.Results The subtractive library of genes transactivated by HCV FTP2 was constructed successfully.The amplified library contains 71 positive clones.Colony PCR shows that 56 clones contain 200～1000 hp inserts.Sequence analysis was performed in 24 clones randomly,and the full length sequences were obtained with bioinformatics method.Altogether 20 coding sequences in total were obtained,consisting of 19 known and 1 un- known.Conclusion The obtained sequences may be target genes transactivated by HCV FTP2,and some genes coding proteins involved in cell cycle regulation,metabolism and cell apoptosis.

Effect of Fusion Protein Cleavage Site Mutations of NDV ZJ1 Strain on Its Fusion Activity / 微生物学通报

NDV strain ZJ1 strain , a highly virulent NDV strain, has been prevalent among the waterfowls in China mainland in the past years. Multi-basic amino acid sequence distribute in the protease cleavage site of F protein of this strain. Recombinant expressing plasmid pCI-FT, was generated by converting multi-basic amino acid sequence of 112, 115, 117 of the protease cleavage site of F_ 0 protein, to the non-basic amino acid sequence characteristic of avirulent NDV strain. The result from co-expression of mutant or parental F protein with homologous HN protein in COS-1 cells revealed that both mutant and parental F protein had fusion activity. The result from co-expression of mutant or parental F protein with homologous HN protein in CEF cells showed that the cleavage activity of mutant F protein was significantly reduced. The study built a foundation for mutagenesis of amino acid sequence of the protease cleavage site of F_ 0 protein at the full-length cDNA clone level, study on factors contributing to virulence and construction of candidate vaccine strain, and so on.

Cloning and bioinformatics analysis of gene F protein binding protein 1 of hepatitis C virus / 解放军医学杂志

Objective To clone and bioinformatically analyze the full-length sequence of F protein binding protein 1 (FBP1). Methods Yeast two-hybrid system was employed to obtain an unknown gene and named it as FBP1.The coding sequence of FBP1 was cloned using molecular biological techniques. Results The coding sequence of FBP1 was cloned successfully. Conclusion FBP1, a cellular protein binding to F protein of HCV, plays an important role in the interaction of virus protein and host cell protein.