Figure 1.
Schematic representation of the domain structure of RHA and the deletions introduced into the linker region.
The proteins are tagged with 6×His at the N-terminus. Shown are the sequence and the relative positions of helices 1 to 6 in the linker region of RHA. Dotted lines represent the deleted helices.
Figure 2.
Ability of purified mutant RHAs to bind and unwind dsRNA in vitro.
(A) Isolation of mutant full-length RHAs. The mutant RHAs containing deletions in the linker region were purified from mammalian cells (HEK 293E), separated by 1D SDS-PAGE, and analyzed by either staining with Coomassie Brilliant Blue R250 (CBR) or probing Western blots with anti-His (WB). Lane M shows a protein size marker with indicated molecular weights in kDa. (B) Diagram of the duplex RNA substrate with one strand 3′-end-labeled with 32pCp. (C) EMSA was carried out to examine the in vitro binding of purified proteins to [32pCp]-labeled synthetic duplex RNA. GST was included as a negative control. Shown is a representative of 3 independent experiments. (D) Helicase activity assay. 10 nM of radioactive duplex RNAs and 150 nM of indicated proteins were incubated at 37°C for indicated time periods in the presence or absence of 1 mM ATP, and then the radioactive RNA strand in single or duplex form was resolved on a 15% native polyacrylamide gel, and visualized using a PhosphorImager. Lane C indicates the migration position of the ssRNA (boiled substrate), while lane N represents the migration position of untreated radioactive duplex RNA. Lanes at time point 0 represent the helicase reactions in the absence of 1 mM ATP. Shown is a representative of 3 independent experiments. (E) Duplex RNA bands in panel D were quantitated using a PhosphorImager, normalized to the results obtained at the time point 0, and presented graphically as percentage. Shown are the mean values ± standard deviations of 3 independent experiments. *, P < 0.05 compared with corresponding values obtained with wild-type RHA.
Figure 3.
Ability of mutant RHAs to interact with HIV-1 RNA in the cell.
293T cells were transfected with SVC21.BH10 and a plasmid expressing either His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cells were cross-linked, lysed, and sonicated. The cell lysates were incubated with Ni-NTA agarose to capture His-tagged protein. RNAs isolated from cell lysates (input) or from nucleoprotein bound to Ni-NTA agarose (precipitate) were subjected to RT-PCR analysis. (A) Western blot of cell lysates was probed with anti-His to detect expression of His-tagged RHA in transfected cells. The expressed 6×His tag peptide alone was not detectable in the Western blot. (B) Western blot of the precipitates was probed with anti-RHA. (C) The input RNA and RNA that was coprecipitated with His-tagged proteins were analyzed by RT-PCR, using primer pair P1-F/R [19] specific to HIV-1 RNA. RT-PCR was performed in the presence (+) or absence (–) of reverse transcriptase.
Figure 4.
Ability of mutant RHAs to stimulate the synthesis of HIV-1 mRNAs.
293T cells were cotransfected with SVC21.BH10 and either a plasmid expressing His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cell lysates and total cellular RNA were prepared and subjected to Western blotting and Northern blotting analysis respectively. (A) Western blots of cell lysates probed with anti-RHA, anti-His, or anti-β-actin. (B) Northern blotting. The total cellular RNA was resolved by electrophoresis on a denaturing 1% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was probed with the [32P]-labeled DNAs that are complimentary to HIV-1 5'-UTR. Ethidium bromide-stained rRNAs (18S and 28S) are included as an RNA loading control. Unspliced (US) ∼ 9.2 kb, singly spliced (SS) ∼ 4.0 kb, and multiply spliced (MS) ∼ 1.8 kb RNAs are indicated. (C) The intensity of RNA bands in panel B representing US, SS, or MS RNAs was quantitated using a PhosphorImager instrument, and are presented graphically. Shown is a representative of 3 independent experiments. (D) The ratio of US RNA to SS+MS RNA in panel B was determined. Shown are the mean values ± standard deviations of 3 independent experiments. *, P < 0.05 compared with values obtained with the 6×His tag alone (lane 1).
Figure 5.
RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species.
Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto GeneScreen Plus membrane, and then probed with [32P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.
Figure 6.
Ability of mutant RHAs to promote the annealing of tRNALys3 to viral RNA.
293T cells were first treated with siRNACon or siRNARHA, and 16 hours later, were cotransfected with SVC21.BH10 and a plasmid expressing either 6×His tag, or His-tagged wild-type or mutant RHAs. 48 hours later, extracellular viruses were purified and cells were lysed. (A) Western blots of cell lysates probed with antibodies to RHA, His tag, or β-actin. (B) Western blots of viral lysates, containing equal amount of CAp24, probed with antibodies to RHA, His tag, CAp24, or RTp66/p51. (C) One nucleotide extension assay (+1 nt extension). Total viral RNA was isolated from purified HIV-1 particles, and tRNALys3 annealed to viral RNA in vivo was extended by 1 nt ([32P]-dCTP), using HIV-1 reverse transcriptase. The extended tRNALys3 products are resolved by denaturing 1D PAGE, and visualized using a PhosphorImager. The control gel represents the +1 nt extension of a DNA primer annealed in vitro to viral RNA downstream of the tRNALys3 binding site, and is used to show that approximately equal amounts of viral RNA were used in each extension reaction. (D) The values of the +1 nt extended tRNALys3 products were quantitated using a PhosphorImager, normalized to the values obtained with virions produced from siRNACon-treated cells (lane 1), and are presented graphically as a percentage. Shown are the mean values ± standard deviations of 3 independent experiments. *, P < 0.05 compared with values obtained with virions produced from cells transfected with a plasmid expressing only His tag (lane 2).
Table 1.
Effect of mutations in the linker region of RHA upon helicase activity in vitro and upon multiple steps in HIV-1 production in the cellsa.
Table 2.
Primers used in fusion PCR to construct recombinant plasmids.