Inhibition of HIV-1 replication by eIF3f

Abstract

Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3' long terminal repeat (3'LTR) region in the viral mRNA. We show that the 3' end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3' end processing of HIV-1 mRNAs.

Fig. 1.

Characterization of the recovered cDNA. (A) Schematic representation of the H2 cDNA fragment. H2 cDNA inserted into retroviral Mo-MuLV vector, pBabeHAZ. ATG¹, start codon at influenza hemaglutinin (HA) tag epitope; ATG², alternative start codon within HA coding sequence; ATG³ start codon for H2(N91-eIF3f)-Zeo. (B) Homology of H2 cDNA N-terminal portion with eIF3f protein. MPN domain, Mrp-1Pad1N-terminal sequence (EX_nHXHX₁₀D). (C) (Left) Western blot analyses (WB) of nuclear and cytoplasmic extracts, from empty vector control TE671 cells (TE) and H2 cellular clone, with an anti-Sh-BLE (zeocin resistance protein). Anti-actin served as a loading control. (Center) detection of both encoded H2 proteins with anti-Sh-BLE or anti-eIF3f antibodies. GAPDH was loading control. (Right) Determination of expression of N91-eIF3f and endogenous eIF3f, using anti-eIF3f antibody in H2 cells; TE cells were used as negative control. (D) Endogenous expression of eIF3f determined using anti-eIF3f antibody. GAPDH was used as a loading control. (E) TE671 cells expressing empty vector control pBabe-HAZ (TE) or H2 cells expressing N91-eIF3f were infected at the indicated multiplicities of infection with VSV-HIV-Puro. Forty-eight hours later, puromycin was added to the medium, and, 5–8 days later, drug-resistant colonies in the plates were counted after Giemsa staining. Results are representative of 3 independent experiments. Error bars represent standard error. (F) Cells tested for susceptibility to a Mo-MuLV vector, VSV-MLV-Neo virus. Transduced drug resistant colonies were scored after Giemsa staining. (G) Cre recombinase was stably introduced into H2 cells by cotransfection with p-Puro. Six puromycin resistant clones were expanded and deletion of H2-Zeo DNA was monitored by PCR. Clones were tested for resistance to VSV-HIV-Neo at a multiplicity of 1 × 10⁻². (H) The H2-Zeo fragment was recovered from H2 cells, cloned into pBabe-HAZ, used to generate retroviral supernatants by transfection, and reintroduced into naïve TE671 cells. Zeocin resistant clones were tested for resistance to transduction after infection with VSV-HIV-Puro. Relative infectivity was determined by comparison to the TE cell line. Results shown are typical of those obtained in 3 independent experiments.

Fig. 2.

Expression of N91-eIF3f and eIF3f and resistance to HIV infection. (A) (Left) Susceptibility of TE671 clones expressing N91-eIF3f-myc (H2-myc) to infection as scored by HIV-Puro colony counting. Clones 1, 2, 3, 4, 5 expressed H2-myc; clones 6 and 7 did not. Clone TEpcDNAcl.1 stably expresses empty vector control. The TE and H2 cell lines were included as negative and positive controls, respectively. Cells were infected at an MOI of 1.5 × 10⁻³ with VSV-HIV-Puro. Forty-eight hours later, puromycin was added to the medium, and, 5–8 days later, resistant colonies in the plates were counted after Giemsa staining. (Right) N91-eIF3f-myc (H2-myc) was transfected into 293T cells, and clones were obtained and tested as for TE671. One empty vector control clone and 2 N91-eIF3f-myc expressing clones were tested for resistance, using the indicated MOIs of VSV-HIV-Puro. (B) Susceptibility of TE671 cells expressing full-length eIF3f-myc. Cellular clones were infected at an MOI of 2.5 × 10⁻³ with VSV-HIV-Puro, and infectivity assayed as in A. (C) (Upper) Expression of transgene protein was assessed in the nucleus and cytoplasm by WB, using an anti c-myc antibody and anti-actin antibody as a loading control. (Lower) Expression of the transgene was determined in total cellular lysates for comparison with endogenous levels of eIF3f, using anti-eIF3f antibody. (D) Full-length eIF3f-myc was stably expressed in 293T cells; eIF3f-myc expression assayed by WB. Cellular clones were tested for resistance at an MOI of 1 × 10⁻³ with VSV-HIV-Puro. A, B, and D are representative of as least 3 independent experiments. (E) RNAi-mediated knockdown of eIF3f and restriction to HIV mediated by N91-eIF3f. TE or H2 cells were transduced with pGIPZ-RNAi construct targeting eIF3f or a scrambled negative control. Lysates of individual clones were analyzed by WB for eIF3f, H2-zeo, or GAPDH as a loading control. TE and H2 lysates were loaded as control. (F) Clones knocked down for eIF3f or scrambled control were tested for susceptibility to infection with HIV-Neo at an MOI of 3 × 10⁻². The average number of puromycin resistance colonies obtained for each set of 3 clones for 3 independent experiments is presented. Error bars represent standard error. (G) TE and H2 cells were infected with VSV-HIV-Puro and selected for viral expression, using increasing amounts of selective drug (puromycin 1 and 5 μg/mL). Detection of H2 cDNA expression by WB as selective pressure for HIV expression increases, using anti-She-BLE and actin as loading control.

Fig. 3.

Analysis of viral block in TE and H2 cell lines. (A) Clones were infected at different multiplicities of VSV-HIV-Puro virus. Total DNA was extracted 7 days after infection and copy number of integrated provirus determined by qPCR. Proviral copy number was normalized per 100 ng of total DNA. Data are representative of 3 independent experiments; error bars represent the standard error. (B) (Left) Analysis of viral mRNA expression. Cytoplasmic mRNA was extracted 7 days after VSV-HIV-Neo infection at the indicated multiplicities. First strand cDNA synthesis and amplification of the target DNA was performed by qPCR, using primers recognizing the neomycin reporter gene. Results were normalized to copies of viral mRNA per copy of GAPDH. (Right) Resistance to infection by HIV-Neo was assessed in parallel. Neomycin was added to the medium 48 h after infection, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results shown are typical of those obtained in 3 independent experiments. (C) (Left) Nuclear and cytoplasmic RNA was extracted 7 days after infection with 10-fold decreasing dilutions of VSV-HIV-TK. After first strand cDNA synthesis, TK and GAPDH cDNA was amplified by PCR. (Right) TE and H2 cells were infected at an MOI of 3 × 10⁻² with HIV-Puro. Seven days after infection; nuclear RNA was extracted and quantified by qRT-PCR, using primers recognizing the puromycin reporter gene. Results are relative to values of GAPDH and the ratio found in TE cells was taken as reference. All data are representative of 3 independent experiments. Errors are standard error of the mean. HI, heat-inactivated.

Fig. 4.

Viral target for H2 mediated resistance. (A) Comparison between different WT HIV and retroviral vectors gene products (arrow) restricted by H2 cells. Amplified dashed box represents the common features: the U3 deleted 3′LTR (dU3LTR), containing the poly(A) signal and a GT rich region, both defining the transcript end. RRE, Rev-responsive element; SFFV, spleen focus forming virus; WPRE, Woodchuck posttranscriptional regulatory element. (B) HIV-puro and a modified HIV-puro with SV40 poly(A) or a BGH poly(A) sequence replacing dU3LTR were transiently transfected into TE or H2 cell lines. Two days after transfection, puromycin was added to the medium, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results are shown as the ratio between the numbers of TE versus H2 resistant colonies and are representative of 3 independent experiments. Error bars represent standard error. (C) In vitro polyadenylation reaction. Poly(A) addition. The ³²P-labeled precleaved RNA substrate was incubated in nuclear extracts from HeLa-CD4-pBabe-HAZ, HeLa-CD4-H2, or HeLa cells and the recombinant eIF3f-GST, N91-eIF3f-GST, and GST proteins, with ATP for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Lanes 10 and 11 are controls of polyadenylation. Precleaved HIV-1 input and HeLa nuclear extracts with 3′dATP or ATP respectively. (D) Poly(A) site cleavage. The ³²P-labeled uncleaved 3′LTR HIV RNA substrate was incubated with nuclear extracts of TE671 or HeLa cells expressing empty vector control (HAZ) or N91-eIF3f (H2) for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. The 3′LTR RNA substrate (HIVwt) or a substrate with a core poly(A) hexamer deletion (HIVΔHex) were incubated with HeLa HAZ extracts for 5′cleavage product control.