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Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research.

ABSTRACT: Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.

SUBMITTER: Takeuchi Y

PROVIDER: S-EPMC2593302 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research.

Takeuchi Yasuhiro Y McClure Myra O MO Pizzato Massimo M

Journal of virology 20081008 24

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01 ...[more]

PMID: 18842727

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Project description:Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif.IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

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Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research.

Publications

Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research.

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