Overexpression of fasciculation and elongation protein zeta-1 (FEZ1) induces a post-entry block to retroviruses in cultured cells

Abstract

Two mutant Rat2 fibroblast cell lines, R3-2 and R4-7, have been previously isolated by a selection for retrovirus resistance. We have now further analyzed the basis of the block to retroviral infection in the R3-2 line. Using Affymetrix GeneChip analysis, several genes were identified as differentially expressed in the mutant R3-2 line compared with the wild-type cells. One of the candidate gene products, FEZ1 (fasciculation and elongation protein zeta-1), a protein kinase C (PKC)zeta-interacting protein homologous to the Caenorhabditis elegans synaptic transport protein UNC-76, was found to be up-regulated >30-fold in the resistant R3-2 line. FEZ1 overexpression in Rat2 cells conferred a potent resistance to infection by genetically marked retroviruses, and the degree of retroviral resistance in both Rat2 fibroblasts and 293T cells tightly correlated with the expression level of FEZ1 transcripts. FEZ1-overexpressing Rat2 cells showed a similar phenotype to that of the mutant R3-2 line: Infection resulted in normal viral DNA synthesis but a reduction in the formation of circular DNA, indicating a block after reverse transcription but before nuclear entry. Partial knockdown of FEZ1 expression in R3-2 by RNA interference (RNAi) significantly reduced the resistance of this line to infection. Thus, our data suggest that FEZ1 overexpression is sufficient to explain the resistant phenotype of R3-2 cells and identify FEZ1 as a new gene capable of causing retrovirus resistance.

Figure 1.

Microarray chip and quantitative RT–PCR data showing genes that are up-regulated in the mutant R3-2 line. FC ratios obtained from the chip analysis are mean of the five or more pairwise comparisons between the mutant R3-2 line and the wild-type Rat2 cells. The FC ratio between the mutant line and the control wild-type cells detected in the RT–PCR analysis are median copy numbers normalized to GAPDH copy numbers for each gene. The RT–PCR results shown for each transcript are typical of those obtained in duplicate using RNA from at least two independent preparations.

Figure 2.

The pattern of expression levels of FEZ1 transcripts correlates with the levels of retroviral resistance in the various Rat2 lines. (A–C) Overexpression of FEZ1 blocks retrovirus infection in Rat2 cells. Cells transduced with the empty vector pcDNA4 or the pcDNA4 vector expressing FEZ1 originated from the mutant R3-2 line, m-FEZ1, were incubated with various amounts of MLV-N-neo (A), MLV-B-neo (B), and HIV-1-puro (C). Cells were selected in medium containing G418 (A,B; Neoviruses) or puromycin (C; HIV-1) and the number of the transduced colonies were then counted after 8–12 d. The wild-type Rat2 cells and the parental R3-2 line were included as negative and positive controls, respectively. Similar results were obtained in at least three independent experiments. (D,E) Quantitative RT–PCR showing the level of FEZ1 expression in Rat2 cells. Cytoplasmic RNA was prepared from the same cell lines used in the transduction assay. The ds cDNA was generated and used as template in RT–PCR assays. Primers unique to each cDNA sequences were used to distinguished the transgene FEZ1 (D) from the total (endogenous and transgene) (E) FEZ1 transcripts. The FC ratio between the Rat2 mutant lines and the control empty vector line (pcDNA4:3) are given as median copy numbers normalized to GAPDH copy numbers obtained in duplicate from two independent RNA preparations.

Figure 3.

The pattern of expression levels of FEZ1 transcripts correlates with the levels of retroviral resistance in the various 293T lines. (A) Overexpression of FEZ1 blocks retrovirus infection in 293T cells. Cells transduced with the empty vector pcDNA4 or the pcDNA4 vector expressing FEZ1 originated from the mutant R3-2 line, m-FEZ1, were incubated with various amounts of HIV-1-puro. Cells were selected in medium containing puromycin and the number of the transduced colonies were then counted after 8–12 d. The wild-type 293T cells and the parental R3-2 line were included as negative and positive controls, respectively. Similar results were obtained in at least three independent experiments. (B,C) Quantitative RT–PCR showing the level of FEZ1 expression in 293T cells. Cytoplasmic RNA was prepared from the same cell lines used in the transduction assay. The ds cDNA was generated and used as template in RT–PCR assays. Primers unique to each cDNA sequences were used to distinguished the transgene FEZ1 (B) from the total (endogenous and transgene) FEZ1 transcripts (C). The FC ratio between the 293T mutant lines and the control empty vector line (pcDNA4:4) are given as median copy numbers normalized to GAPDH copy numbers. Two independent RNA preparations were used in the analysis.

Figure 4.

Quantitative RT–PCR indicating the localization of the viral block in FEZ1 stable line. One of the Rat2 line stably expressing FEZ1 (m-FEZ1:8), the control empty vector pcDNA4:5 line, the wild-type Rat2 cells, and the parental R3-2 were infected with either undiluted (un) or 10-fold serially diluted (10×, 100×) ecotropic MLV-GFP. As a negative control, uninfected cells (indicated as 0 after the sample name) for each sample were included in the experiment. Total DNA was isolated at 24 and 36 h after infection and the amount of viral DNA synthesized in the infected cells was measured by RT–PCR. Using primers specific to GFP sequences, minus strand strong stop (MSS) DNA or LTR–LTR junction, the amounts of the total viral DNA (A), MSS DNA (B), or circular viral DNA in the nucleus (C) were determined, respectively. The DNA copy number for each sample was normalized to the copy number of the internal control GAPDH. Each DNA sample was assayed in duplicates at the minimum of three different time points.

Figure 5.

RNAi to FEZ1 reduces the resistance of the mutant R3-2 line. Wild-type or mutant R3-2 cells were transfected with three independent short-interfering RNAs (indicated as A, B, or C) on two consecutive days with equal amounts of RNA duplexes and subsequently seeded and infected with various amounts of MLV-N-neo. Cells were selected in medium containing G418, and the number of the transduced colonies was then counted after 8–12 d. The wild-type Rat2 cells and a non-specific GFP duplex were included as negative controls. Similar results were obtained in at least three independent experiments.