Ring finger protein ZIN interacts with human immunodeficiency virus type 1 Vif

Abstract

Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-kappaB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.

FIG. 1.

Sequence analysis of Triad 3 and ZIN. Deduced protein sequences of the 488-amino-acid ZIN and the 218-amino-acid Triad 3 sequences are shown. These sequences were determined in the present study. The GenBank accession no. for the previously published ZIN protein sequence is NP_061884. Triad 3 amino acid residues that are identical to the ZIN sequence are marked with asterisks. The two amino acid residues different from the published ZIN sequence are circled (see text). RLDs are enclosed by open boxes. A proline-rich domain (PRD) is underlined.

FIG. 2.

Triad 3/ZIN mRNA is expressed in permissive and nonpermissive cells. Northern blot hybridization of total RNA extracted from nonpermissive HUT-78, H9, PBLs, macrophages, and A3.01 cells (lanes 1 to 5) and permissive 293T, CEM-ss, Cos-7, and HeLa cells (lanes 6 to 9). The filter was first probed with ³²P-labeled Triad 3 cDNA (A) and then stripped and reprobed with ³²P-labeled GAPDH cDNA (B).

FIG. 3.

Vif binds ZIN in vitro. Western blot analysis of total protein in pFlag-ZIN-transfected 293T cell lysate or protein pulled down by GST protein-bound glutathione Sepharose CL-4B beads or by GST-Vif fusion protein-bound beads. The same filter was analyzed by Western blot successively probed with monoclonal anti-Flag antibody (A), anti-GST antibody (B), and polyclonal anti-Vif antiserum (C).

FIG. 4.

Coimmunoprecipitation of Vif and Triad 3/ZIN. (A, B, and C) Western blot analysis of total protein (A and B) and protein immunoprecipitated by HA-specific rabbit polyclonal antiserum (C) from transfected 293T cell lysate probed with HA-specific rabbit polyclonal antiserum (A) or monoclonal mouse anti-Myc antibody (B and C). (D) Total protein and protein immunoprecipitated with mouse anti-Flag antibody from transfected 293T cells analyzed by Western blot probed with polyclonal rabbit anti-Vif antiserum. Plasmid DNAs used in different transfection treatments are labeled on top of the lanes.

FIG. 5.

Virus produced in cells overexpressing ZIN has reduced single-cycle infectivity. HIV virus stocks were produced from 293T cells cotransfected with pNL4.3 (or pNL4.3(Δvif)) and either pFlag or pFlag-ZIN. HeLa-CD4-LTR-β-gal cells were infected with a 100-μl inoculum made of 10-fold dilutions of 40 ng of p24 of each of the four virus stocks in 48-well plates. At 30 h postinfection, cells were fixed and stained as described previously. Blue cells were counted in each well infected at a virus dilution of 10⁻² (0.4 ng of p24/well) in which there are minimum of 80 blue cells. Error bars were obtained from infections performed in nine replica wells.

FIG. 6.

Virus produced in cells overexpressing ZIN has impaired reverse transcription in newly infected cells: Levels of HIV-1 strong-stop DNA (ssDNA) (A and C) and U5/Gag DNA (B and D) in 10⁶ HUT-78 cells infected with 10 ng of p24 of HIV virus stocks 16 and 24 h postinfection. Virus stocks were produced from 293T cells cotransfected with pNL4.3 and pFlag or pFlag-ZIN (A and B) or pNL4.3(Δvif) and pFlag or pFlag-ZIN (C and D). Viral DNA synthesis was quantified by real-time PCR. DNA copy number values represent the average of four PCR measurements from duplicate infected wells.