title: Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.
abstract: This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to transfect the patients' PMBCs with the above four clones. The phylogenetic tree of the C2V3 segment of the Env gene showed that a significant gene cluster was formed by all of the chimeric full-length HNXX1306 clones, and the bootstrap value for this cluster was 97.5%. Patients' PBMCs could be infected by 1306N6, 1306N13 and 1306N22 chimeric full-length clones. The CRF07_BC subtype (6889-7407 nucleotide residues of HXB2) is one of the most prevalent epidemic HIV-1 virus strains among the MSM population. The full-length chimeric molecular clone pNL4-3/07BCLTR may significantly improve the in vitro infectivity of the CRF07_BC strain.
authors.affiliation: Youth League Committee, The Second Affiliated Hospital of Kunming Medical University, Kunming 650500, PR China.
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Jiang
authors.affiliation: Department of Internal Medicine-Cardiovascular, The Second Affiliated Hospital of Kunming Medical University, Kunming 650500, PR China.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Bai
authors.affiliation: International College, Kunming Medical University, Kunming 650500, PR China.
authors.forename: Fan-Wei
authors.initials: FW
authors.lastname: Qu
authors.affiliation: Students' Affairs Division, Kunming Medical University, Kunming 650500, PR China.
authors.forename: Hua
authors.initials: H
authors.lastname: Ma
authors.affiliation: Public Health Hospital, Kunming Medical University, Kunming 650500, PR China. Electronic address: rs_jiang2015@126.com.
authors.forename: Run-Sheng
authors.initials: RS
authors.lastname: Jiang
authors.affiliation: Public Health Hospital, Kunming Medical University, Kunming 650500, PR China.
authors.forename: Bao-Sheng
authors.initials: BS
authors.lastname: Shen
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D018451:Homosexuality, Male; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008297:Male; D016133:Polymerase Chain Reaction; D059386:Reverse Genetics
keywords: CRF07_BC; Full-length cDNA clone; HIV-1; Infectious molecular clone; Long terminal repeat; Men who have sex with men