Inhibition of HIV-1 Gene Expression by a Fragment of hnRNP U Susana T. Valente, Stephen P. Goff  Molecular Cell  Volume 23, Issue 4, Pages 597-605 (August 2006) DOI: 10.1016/j.molcel.2006.07.021 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Resistance of H1-Expressing Cells to Virus Infection (A) TE or TE.H1 cells were infected at low multiplicity of infection with VSV-HIV-Puro, and, 48 hr later, puromycin was added to the medium. Five to eight days later, resistant colonies in the plates were counted after Giemsa staining. Results are representative of three independent experiments, and errors bars represent standard error of the mean. (B) Cells tested for VSV-MLV-Neo resistance. (C) Cre recombinase was stably introduced into TE.H1 cells by cotransformation with pNeo. Seven neomycin-resistant clones were expanded and tested for resistance to VSV-HIV-Puro. Deletion of H1-Zeo DNA was monitored by PCR; the excision was successful in clones TE.H1 cre Cl. 1, 2, 3, 4, and 5 and not in clones Cl. 6 and 7. Clones were tested for resistance with HIV-Puro with moi 3 × 10−3. (D) The H1-Zeo fragment was recovered from TE.H1 cells, cloned into pBabe-HAZ, packaged, and reintroduced into TE cells. The TE.H1 clones 1, 2, 3, 4, 5, 7, and 8 expressed H1-Zeo DNA, but TE.H1 clones 9 and 10 did not. Clones were tested for resistance upon infection by VSV-HIV-Puro at moi 3 × 10−3. Relative infectivity was determined by normalizing the number of resistant colonies to that obtained with TE cell line. Molecular Cell 2006 23, 597-605DOI: (10.1016/j.molcel.2006.07.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Schematic Representation of the cDNA Fragment Recovered from TE.H1 Cells (A) Schematic representation of the cDNA fragment recovered from TE.H1 cells. ATG, start codon; TGA, stop codon in 5′UTR; H1(N-86-hnRNPU)-Zeo, ORF of 86 amino acids of hnRNP U fused to zeocin-resistance gene. (B) Homology of H1 cDNA N-terminal portion with hnRNP U protein. SAP, DNA binding region; RGG, RNA binding region. (C) Expression of the fusion protein in TE or TE.H1 cells was determined by immunoblotting with an anti-Sh BLE (zeocin-resistance protein), using anti-actin as a loading control. (D) Overexpression of N-86-hnRNPU is sufficient to restrict HIV-1 expression. Several cellular clones of TE cells stably expressing N-86-hnRNPU-myc were infected at low multiplicity of infection with VSV-HIV-Puro. Cells were selected in puromycin-containing medium, and, 5–8 days later, puromycin-resistant colonies were scored after Giemsa staining. The TE and TE.H1 cell lines were included as negative and positive controls, respectively. (E) Several clones of 293T cells overexpressing N-86-hnRNPU-myc or the empty vector control pcDNA4 were tested for their susceptibility to HIV-Puro infection as in (D). The level of N-86-hnRNPU-myc expressed by the clones was assessed by immunoblotting with an anti-myc antibody, using anti-tubulin as a loading control. Molecular Cell 2006 23, 597-605DOI: (10.1016/j.molcel.2006.07.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of Viral Block in TE and TE.H1 Cell Lines (A) (Top) Clones were infected at different multiplicities of VSV-HIV-Puro virus. Total DNA was extracted 7 days postinfection, and copy number of integrated provirus was determined by qPCR using oligonucleotides for the puromycin-resistance gene. Proviral copy number normalized per 100 ng of total DNA. (Bottom) In parallel, clones were tested for VSV-HIV-Puro resistance. Puromycin was added to the medium 48 hr postinfection, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results shown are typical of those obtained in three independent experiments. (B) (Top) Analysis of viral mRNA expression. Cytoplasmic mRNA was extracted 7 days post VSV-HIV-Neo infection at the indicated multiplicities. First-strand cDNA synthesis and amplification of the target DNA were performed by qPCR using primers recognizing the neomycin reporter gene. Results were normalized to copies of viral mRNA per copy of GAPDH. Data are representative of three independent experiments, each with duplicate PCR. Errors are standard error of the mean. (Bottom) Resistance to infection by HIV-Neo was assessed in parallel. (C) Nuclear and cytoplasmic RNA was extracted 7 days postinfection with 10-fold decreasing dilutions of VSV-HIV-TK. After first-strand cDNA synthesis, TK and GAPDH cDNA were amplified by PCR. HI, heat inactivated. (D) Analysis of different viral messages blocked by N-86-hnRNPU. The virus VSV/pNL4-3/GFP+/Env− was used to infect TE or TE.H1 cells at different multiplicities of infection. Twelve hours postinfection, the cytoplasmic and nuclear mRNA fractions were collected and reverse transcribed, and the amounts of single-spliced, unspliced, and total viral RNA were determined by qPCR. The fold changes were normalized first to GAPDH detection threshold value (ct) for each sample, and then TE.H1 was compared to TE values. Molecular Cell 2006 23, 597-605DOI: (10.1016/j.molcel.2006.07.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Replication of Wt HIV-1 (pNL4-3) in Cells Expressing H1 (Left) Production of virions. HeLa-CD4 and HeLa-CD4 H1-expressing clones were inoculated with serial dilutions of wild-type HIV-1. Virus in culture medium was quantified by measurement of virion-associated RT activity on an exogenous homopolymer template. Supernatants were assayed for viral particle production on successive days postinfection. Results for the highest viral inoculation are presented. (Right) Viral load measured by Tat transactivation of an HIV-1 LTR-Luciferase reporter gene in PL11 cells. Viral supernatants on day 3 postinfection (left) were collected and used to infect PL11 cells. Virus was allowed to amplify in these permissive cells, and luciferase levels were scored 48 hr later. Results shown are typical of those obtained in three independent experiments. Molecular Cell 2006 23, 597-605DOI: (10.1016/j.molcel.2006.07.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Viral Target for H1 Resistance (A) Comparison between different retroviral vector gene products (arrow) restricted by TE.H1. Amplified dashed box represents the following common features: the 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) pPuro, HIV-Puro, and a modified HIV-Puro with SV40 poly(A) or a BGH poly(A) sequence replacing the dU3LTR were transiently transfected into TE or TE.H1 clones. 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 number of TE- and TE.H1-resistant colonies and are representative of three independent experiments. Molecular Cell 2006 23, 597-605DOI: (10.1016/j.molcel.2006.07.021) Copyright © 2006 Elsevier Inc. Terms and Conditions