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Constructing eukaryotic expressing vector of pMT/Bip/V5-His A-HIV gp120 and transfecting S2 cells to establish stable gp120-expressing S2 cell line. The gp120 gene of HIV CRF07-BC epidemic in China was amplified by polymerase chain reaction from the recombinant vector PRSV-gp120 and confirmed by DNA sequence analysis. The DNA fragment of HIV gp120 was digested with the restriction endonucleases NcoI and XhoI, then inserted into eukaryotic expressing vector pMT/BiP/V5-His A. A selection vector containing the streptomyces griseochromogenes bsd gene conferring blasticidin resistance was cotransfected into drosophila S2 cells by Cellfectin II reagent. The stable cell line was established following repeated screening by blasticidin. HIV gp120 was purified by a Ni-NTA affinity column followed by molecular sieve chromatography. Recombinant protein was characterized by SDS-PAGE, Western blot and ELISA. The eukaryotic expressing vector pMT/BiP/V5-His A-HIV gp120 was constructed, stable expressing cell line was established, and protein was expressed and purified successfully. This result will contribute to functional study of gp120 and vaccine design against AIDS.

Objective: To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors., Methods: HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method., Results: No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner., Conclusion: We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.

Objective: To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6., Methods: The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed., Results: There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity., Conclusion: The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.

Objective: To investigate the biological characteristics of the HIV-1 isolates circulating in China and to define the association of these properties with env V3 loop sequence variability., Methods: Primary viruses were isolated from fresh peripheral blood mononuclear cells (PBMCs) using the traditional co-culture method and their capacity of inducing syncytium was tested in MT-2 cells; meanwhile, their coreceptor usage was determined with GHOST-cell lines which stably express CD4 and the chemokine receptor CCR5 or CXCR4. Furthermore, HIV-1 V3 and its flanking region sequences were amplified by nest-polymerase chain reaction (nest-PCR) and sequenced. A GCG software was used to translate the DNA sequences into polypeptide sequences., Results: Five primary viral strains were isolated from 3 different regions in China. The isolates LTG0213 and LTG0214 induced syncytia in MT-2 cells and used CXCR4 as coreceptor. The isolates XJN0021, XJN0091, and SHXDC0041 did not induce syncytia and used CCR5 as coreceptor. There were obvious differences between X4/SI and R5/NSI viruses in env V3 loop sequences. A consensus motif at the positions 8, 11, 18, and 25 in V3 loop was identified as follows: a sequence as "8-TXXS/GXXXXXXR/QXXXXXXE/D-25" will predict the usage of CCR5 coreceptor; a sequence replacing these positions with basic amino acids (except position 25) will very likely predict the usage of CXCR4 coreceptor., Conclusion: The biological characteristics of HIV isolates are linked to env V3 loop sequence variability: introducing basic amino acids (or translating from acidic amino acids into neutral amino acids) at the positions 8, 11, 18, and 25 in V3 loop will change viral strain's biological phenotype from NSI/CCR5 to SI/CXCR4. The biological phenotype of HIV-1 can be predicted with V3 loop sequence analysis.

The first step of viral infection is virus fusion with target membrane. The process is induced by fusion peptide located in glycoprotein on virus membrane. Fusion peptide exploited after interaction with receptors and penetrates into target cells membrane. Lipid molecules surrounding peptides are rearranged and membrane fusion occurs through the formation of a intermediate state. This review introduces the research advances of viral fusion peptides and its mechanism of penetrating into membrane.