1. Volume 36, Issue 2, Pages 279-289 (October 2009)
HIV-1 mRNA 3′ End Processing Is Distinctively Regulated by eIF3f, CDK11, and Splice Factor 9G8 
Susana T. Valente, Greg M. Gilmartin, Krishnan Venkataraman, Gloria Arriagada, Stephen P. Goff 
Molecular Cell 
Volume 36, Issue 2, Pages (October 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 N91-eIF3f Restricts Wild-Type HIV-1 Replication
(A) One HeLa-CD4 cell clone expressing empty vector control pBabe-HAZ, and three independent H2 cDNA-expressing clones were tested for susceptibility to infection with VSV-HIV-Puro.
(B) Replication of WT HIV-1 in resistant cell lines. HeLa-CD4-HAZ and HeLa-CD4 H2-expressing clones were inoculated with serial dilutions of WT HIV-1. Virus in culture medium was quantified by measurement of virion-associated RT activity. Supernatants were harvested for a period of 10 days and assayed for viral particle production. Cells were split at days 6 and 9 postinfection.
(C) Viral load measured by Tat transactivation of an HIV-1 LTR-luciferase reporter gene in HeLa-CD4-LTR-luciferase indicator cells. Viral supernatants on day 3 postinfection (B) were collected and used to infect indicator cells. Luciferase levels were scored 48 hr later. Results shown are typical of those obtained in three independent experiments.
(D) Cell-cycle analysis. Histograms of TE and H2 cell lines stained with propidium iodide and digestion with RNase. Numbers at the top represent percentages of cells in G1 early S phase; S phase or late S-G2-M.
(E) Mitochondrial metabolic activity. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on TE and H2 cell lines. Absorbance at 595 nm reports the ability of mitochondrial dehydrogenase enzyme in viable cells to cleave tetrazolium rings of the yellow MTT and form dark blue formazan crystals.
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3. Figure 2 Processing Efficiency of HIV-1 Poly(A) Site In Vitro
(A) Schematic depiction of the HIV-1 pre-mRNA 3′ end-processing substrates, and expected products of in vitro cleavage and polyadenylation reactions. The cleavage site, the dinucleotide -CA-, lies 25 nt downstream from the AAUAAA. The SP6 transcripts used in the polyadenylation and cleavage assays are depicted below.
(B) Poly(A) site cleavage. 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). The RNA products were resolved on a denaturing gel. The 3′LTR RNA substrate (HIVwt) or a substrate with a core poly(A) hexamer deletion (HIVΔHex) was incubated with HeLa HAZ extracts for 5′ cleavage product control.
(C) (Left) Poly(A) site cleavage using HeLa HAZ or HeLa H2 nuclear extracts and the recombinant eIF3f-GST, N91-eIF3f-GST, and GST proteins. (Right) Densitometric plot of the cleavage activity by measurement of 5′ cleaved product for each nuclear extract above background.
(D) (Left) Poly(A) site cleavage. 32P-labeled RNAs containing the 3′ poly(A) sites of HIV-1; Drosophila melanogaster Notch1; Adenovirus L3; SV40 early genes; and the noncanonical human VTI1B, Nab1, and GluL genes were incubated in nuclear extracts of HeLa-CD4-HAZ and HeLa-CD4-H2 cells. (Top right) Notch1, Ad-L3, and SV40 poly(A) sites deleted or not of the core hexamer were incubated with HeLa-CD4-HAZ extracts and included as a control. (Bottom right) Densitometric plot of the cleavage activity.
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4. Figure 3 Processing Efficiency of HIV-1 Poly(A) Site In Vivo
(A) Depiction of the retroviral vector used for functional studies. RRE, Rev responsive element; SFFV, spleen focus forming virus; WPRE, woodchuck posttranscriptional regulatory element; PuroR, puromycin resistance gene.
(B) An HIV-puro construct (1), HIV-puro constructs with BGH (4) or SV40 (5) poly(A)s sequences replacing dU3LTR, and constructs in which BGH (2) and SV40 poly(A)s (3) were placed downstream from the 3′LTR were transiently transfected into (left) TE or H2 cell lines or (right) TE-pcDNA4 and TE-eIF3f cells. Two days after transfection, puromycin was added to the medium, and 5–8 days later, resistant colonies were scored. Results are shown as the ratio between the numbers of TE versus H2-resistant colonies, representative of three independent experiments. Error bars represent standard error.
(C) Assessment of poly(A) usage in a poly(A) tandem construct in vivo. HIV-Puro vectors containing the BGH poly(A) (left) or the SV40 poly(A) (right) downstream from the 3′LTR poly(A) site were stably expressed with very low concentration of puromycin, to avoid loss of N91-eIF3f overexpression, in TE671 cells expressing full-length eIF3f or empty vector control. mRNA was extracted from these cells and cDNA made using poly dT. The ratio of 3′LTR poly(A) site versus heterologous poly(A) site usage was determined by qPCR using the indicated primer pair, A with B and A with C (left) or A with B and A with D (right) for each cell line. Experiment is representative of three independent experiments.
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5. Figure 4
eIF3f, 9G8, and CDK11
TE671 cells were transiently transfected with DNAs encoding 9G8-N-FLAG, CDK11-C-FLAG, eIF3f-myc, or pcDNA3.1 (as control) as indicated for 48 hr. Five hundred micrograms of cell lysates were immunoprecipitated with M2 anti-FLAG, and protein complexes were resolved by SDS-page followed by immunoblotting with the anti-eIF3f antibody. The blots were subsequently stripped and reprobed with anti-M2 FLAG antibody. Five percent of input lysate was analyzed by immunoblotting using the same antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Restriction of HIV-1 Mediated by CDK11 and 9G8
(A) Susceptibility of TE671 clones expressing CDK11-C-FLAG to infection as scored by HIV-Puro colony counting. The TE and H2 cell lines were included as negative and positive controls, respectively. Cells were infected at a moi of 1.5 × 10−3 with VSV-HIV-Puro; 48 hr later, puromycin was added to the medium; and 5–8 days later, resistant colonies in the plates were counted.
(B) H2 clone or H2 clones stably expressing CDK11-C-FLAG were tested for HIV-Puro susceptibility as above.
(C) TE and H2 cells were stably transfected with 9G8-N-FLAG, and the populations were scored for HIV resistance as in (A). Results shown are typical of those obtained in three independent experiments. Error bars represent standard error. pop, cell population.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Processing Efficiency of HIV-1 Poly(A) Site
(A) Schematics of the 3′LTR region of HIV-1. The poly(A) site hexamer is indicated, and upstream is highlighted the 9G8 potential binding site. The gray arrows denote the primers used to make the probes for RNA-EMSA, containing or not the mutations in the 9G8 site.
(B) Gel mobility shift analysis of HeLa-HAZ and HeLa-N91-eIF3f/RNA complexes. Nuclear extracts were incubated with 32P-labeled RNAs, and the RNA/protein complexes were resolved on a nondenaturing gel. WT, wild-type 9G8 sequence; mut9G8, mutated 9G8 sequence.
(C) Gel mobility shift assay using recombinant GST-9G8 with the same probes as in (B).
(D) (Left) Poly(A) site cleavage. 32P-labeled uncleaved substrates of WT 3′LTR HIV RNA or 3′LTR with the indicated (A) point mutations were incubated with nuclear extracts of HeLa HAZ. The RNA products were isolated and resolved on a denaturing gel. (Right) Densitometric plot of the cleavage activity for each RNA substrate by measurement of the 5′ cleaved product above background.
(E) Susceptibility of TE671 cells to infection by WT-HIV-Puro or mut9G8-HIV-Puro as scored by puromycin-resistant colony count. Cells were infected at a multiplicity of 2 × 10−3 with VSV-pseudotyped virus either carrying the 9G8 mutation or not; 48 hr later, puromycin was added to the medium; and 5–8 days later, resistant colonies in the plates were counted. Error bars represent standard error.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Proposed Model for N91-eIF3f Inhibition of HIV-1 mRNA 3′ End Cleavage
Schematic drawing of the pre-mRNA 3′ end-processing complexes and cis elements. CPSF recognizes the poly(A) signal, whereas CstF recognizes the downstream sequence element (DSE). The CFIm preferentially binds to the sequence UGUAN (N = A > U ≥ C/G) and enhances the binding of CPSF. The HIV-1 3′LTR contains a sequence related to the consensus-binding site of 9G8. 9G8 directly interacts in vivo and in vitro with CDK11. CDK11 interacts with eIF3f, and therefore CDK11 may serve to link eIF3f and mRNA 3′ end processing. 9G8 also specifically interacts with the CFIm and is thought to bridge regulation of alternative splicing and 3′ end processing. Overexpression of eIF3f or N91-eIF3f may disrupt the eIF3f-CDK11-9G8 interactions and, in turn, would prevent the proper modification of 9G8/CFIm/CPSF required for efficient HIV poly(A) site recognition. Poly(A) regions that do not contain 9G8 binding sequences would not be perturbed by the excess of eIF3f or N91-eIF3f, as 9G8 would not be involved in the overall process. Poly(A) polymerase (PAP), cyclin-dependent kinase CDK11, SR protein 9G8.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions